Identification of virulence associated regions rd1 and rd5 leading to improve vaccine of m. bovis bcg and m. microti

ABSTRACT

The present invention relates to a strain of  M. bovis  BCG or  M. microti,  wherein said strain has integrated part or all of the RD1 region responsible for enhanced immunogenicity of the tubercle bacilli, especially the ESAT-6 and CFP-10 genes. These strains will be referred as the  M bovis  BCG::RDI or  M. microti ::RD1 strains and are useful as a new improved vaccine for preventing tuberculosis and as a therapeutical product enhancing the stimulation of the immune system for the treatment of bladder cancer. These strains are also useful for the expression and presentation of heterologous antigens and molecule that are of therapeutic or prophylactic interest.

Virulence associated regions have been sought for a long time inMycobacterium. The present invention concerns the identification of 2genomic regions which are shown to be associated with a virulentphenotype in Mycobacteria and particularly in M. tuberculosis. Itconcerns also the fragments of said regions.

One of these two regions are known as RD5 as disclosed in MolecularMicrobiology (1999), vol. 32, pages 643 to 655 (Gordon S. V. et al.).The other region named RD1-2F9 spans the known region RD1 as disclosedin Molecular Microbiology (1999), vol. 32, pages 643 to 655 (Gordon S.V. et al.). Both of the regions RD1 and RD5 or at least one of them areabsent from the vaccine strains of M. bovis BCG and in M. microti,strains found involved and used as live vaccines in the 1960's.

Other applications which are encompassed by the present invention arerelated to the use of all or part of the said regions to detect virulentstrains of Mycobacteria and particularly M. tuberculosis in humans andanimals. The region RD1-2F9 and RD5 are considered as virulence markersunder the present invention.

The recombinant Mycobacteria and particularly M. bovis BCG aftermodification of their genome by introduction of all or part of RD1-2F9region and/or RD5 region in said genome can be used for the immunesystem of patients affected with a cancer as for example a bladdercancer.

The present invention relates to a strain of M. bovis BCG or M microti,wherein said strain has integrated all or part of the region RD1-2F9responsible for enhanced immunogenicity to the tubercle bacilli,especially the genes encoding the ESAT-6 and CFP-10 antigenes. Thesestrains will be referred to as the M. bovis BCG::RD1 or M. microti::RD1strains and are useful as a new improved vaccine for prevention oftuberculosis infections and for treating superficial bladder cancer.

Mycobacterium bovis BCG (bacille Calmette-Guérin) has been used since1921 to prevent tuberculosis although it is of limited efficacy againstadult pulmonary disease in highly endemic areas. Mycobacterium microti,another member of the Mycobacterium tuberculosis complex, was originallydescribed as the infective agent of a tuberculosis-like disease in voles(Microtus agrestis) in the 1930's (Wells, A. Q. 1937. Tuberculosis inwild voles. Lancet 1221 and Wells, A. Q. 1946. The murine type oftubercle bacillus. Medical Research council special report series259:1-42.). Until recently, M. microti strains were thought to bepathogenic only for voles, but not for humans and some were even used asa live-vaccine. In fact, the vole bacillus proved to be safe andeffective in preventing clinical tuberculosis in a trial involvingroughly 10,000 adolescents in the UK in the 1950's (Hart, P. D. a., andI. Sutherland. 1977. BCG and vole bacillus vaccines in the prevention oftuberculosis in adolescence and early adult life. British MedicalJournal 2:293-295). At about the same time, another strain, OV166, wassuccessfully administered to half a million newborns in Prague, formerCzechoslovakia, without any serious complications (Sula, L., and I.Radkovsky. 1976. Protective effects of M. microti vaccine againsttuberculosis. J. Hyg. Epid. Microbiol. Immunol. 20:1-6). M. microtivaccination has since been discontinued because it was no more effectivethan the frequently employed BCG vaccine. As a result, improved vaccinesare needed for preventing and treating tuberculosis.

The problem for attempting to ameliorate this live vaccine is that themolecular mechanism of both the attenuation and the immunogenicity ofBCG is still poorly understood. Comparative genomic studies of all sixmembers of the M. tuberculosis complex have identified more than 140genes, whose presence is facultative, that may confer differences inphenotype, host range and virulence. Relative to the genome of theparadigm strain, M. tuberculosis H37Rv (S. T. Cole, et al., Nature 393,537 (1998)), many of these genes occur in chromosomal regions that havebeen deleted from certain species (RD1-16, RvD1-5), M. A. Behr, et al.,Science 284, 1520 (1999); R. Brosch, et al., Infection Immun. 66, 2221(1998); S. V. Gordon, et al., Molec Microbiol 32, 643 (1999); H.Salamon, et al, Genome Res 10, 2044 (2000), G. G. Mahairas et al, J.Bacteriol. 178, 1274 (1996) and R. Brosch, et al., Proc Natl Acad SciUSA 99, 3684 (2002).

In connection with the invention and based on their distribution amongtubercle bacilli and potential to encode virulence functions, RD1,RD3-5, RD7 and RD9 (FIG. 1A, B) were accorded highest priority forfunctional genomic analysis using “knock-ins” of M. bovis BCG to assesstheir potential contribution to the attenuation process. Clones spanningthese RD regions were selected from an ordered M. tuberculosis H37Rvlibrary of integrating shuttle cosmids (S. T. Cole, et al, Nature 393,537 (1998) and W. R. Bange, et al, Tuber. Lung Dis. 79, 171 (1999)), andindividually electroporated into BCG Pasteur, where they inserted stablyinto the attB site (M. H. Lee, et al, Proc. Natl. Acad. Sci. USA 88,3111 (1991)).

We have uncovered that only reintroduction of all or part of RD1-2F9 ledto profound phenotypic alteration. Strikingly, the BCG::RD1 “knock-in”grew more vigorously than BCG controls in immuno-deficient mice,inducing extensive splenomegaly and granuloma formation.

RD1 is restricted to the avirulent strains M. bovis BCG and M. microti.Although the endpoints are not identical, the deletions have removedfrom both vaccine strains a cluster of six genes (Rv3871-Rv3876) thatare part of the ESAT-6 locus (FIG. 1A (S. T. Cole, et al., Nature 393,537 (1998) and F. Tekaia, et al., Tubercle Lung Disease 79, 329 (1999)).

Among the missing products are members of the mycobacterial PE (Rv3872),PPE (Rv3873), and ESAT-6 (Rv3874, Rv3875) protein families. Despitelacking obvious secretion signals, ESAT-6 (Rv3875) and the relatedprotein CFP-10 (kv3874), are abundant components of short-term culturefiltrate, acting as immunodominant T-cell antigens that induce potentTh1 responses (F. Tekaia, et al., Tubercle Lung Disease 79, 329 (1999);A. L. Sorensen, et al, Infect. Immun. 63, 1710 (1995) and R. Colangelli,et al., Infect. Immun. 68,990 (2000)).

In summary, we have discovered that the restoration of RD1-2F9 to M.bovis BCG leads to increased persistence in immunocompetent mice. The M.bovis BCG::RD1 strain induces RD1-specific immune responses of theTh1-type, has enhanced immunogenicity and confers better protection thanM. bovis BCG alone in animal models of tuberculosis. The M. bovisBCG::RD1 vaccine is significantly more virulent than M. bovis BCG inimmunodeficient mice but considerably less virulent than M.tuberculosis.

In addition, we show that M. microti lacks a different but overlappingpart of the RD1 region (RD1^(mic)) to M. bovis BCG and our resultsindicate that reintroduction of RD1-2F9 confers increased virulence ofBCG::RD1 in immunodeficient mice. The rare strains of M. microti thatare associated with human disease contain a region referred to asRD5^(mic) whereas those from voles do not. M. bovis BCG vaccine could beimproved by reintroducing other genes encoding ESAT-6 family membersthat have been lost, notably, those found in the RD8 and RD5 loci of M.tuberculosis. These regions also code for additional T-cell antigens.

M. bovis BCG::RD1 could be improved by reintroducing the RDS and RD5loci of M. tuberculosis.

M. bovis BCG vaccine could be improved by reintroducing andoverexpressing the genes contained in the RD1, RD5 and RD8 regions.

Accordingly, these new strains, showing greater persistence and enhancedimmunogenicity, represent an improved vaccine for preventingtuberculosis and treating bladder cancer.

In addition, the greater persistence of these recombinant strains is anadvantage for the presentation of other antigens, for instance from HIVin humans and in order to induce protection immune responses. Thoseimproved strains may also be of use in veterinary medicine, for instancein preventing bovine tuberculosis.

Description

Therefore, the present invention is aimed at a strain of M. bovis BCG orM. microti, wherein said strain has integrated all or part of theRD1-2F9 region as shown in SEQ ID No 1 responsible for enhancedimmunogenicity to the tubercle bacilli. These strains will be referredto as the M. bovis BCG::RD1 or M. microti::RD1 strains.

In connection with the invention, “part or all of the RD1-2F9 region”means that the strain has integrated a portion of DNA originating fromMycobacterium tuberculosis or any virulent member of the Mycobacteriumtuberculosis complex (M. africanum, M. bovis, M. canettii), whichcomprises at least one, two, three, four, five, or more gene(s) selectedfrom Rv3861 (SEQ ID No 4), Rv3862 (SEQ ID No 5), Rv3863 (SEQ ID No 6),Rv3864 (SEQ ID No 7), Rv3865 (SEQ ID No 8), Rv3866 (SEQ ID No 9), Rv3867(SEQ ID No 10), Rv3868 (SEQ ID No 11), Rv3869 (SEQ ID No 12), Rv3870(SEQ ID No 13), Rv3871 (SEQ ID No 14), Rv3872 (SEQ ID No 15,mycobacterial PE), Rv3873 (SEQ ID No 16, PPE), Rv3874 (SEQ ID No 17,CFP-10), Rv3875 (SEQ ID No 18, ESAT-6), Rv3876 (SEQ ID No 19), Rv3877(SEQ ID No 20), Rv3878 (SEQ ID No 21), Rv3879 (SEQ ID No 22), Rv3880(SEQ ID No 23), Rv3881 (SEQ ID No 24), Rv3882 (SEQ ID No 25), Rv3883(SEQ ID No 26), Rv3884 (SEQ ID No 27) and Rv3885 (SEQ ID No 28). Theexpression “a portion of DNA” means also a nucleotide sequence or anucleic acid or a polynucleotide. The expression “gene” is referredherein as the coding sequence in frame with its natural promoter as wellas the coding sequence which has been isolated and framed with anexogenous promoter, for example a promoter capable of directing highlevel of expression of said coding sequence.

In a specific aspect, the invention relates to a strain of M. bovis BCGor M. microti wherein said strain has integrated at least one, two,three or more gene(s) selected from Rv3867 (SEQ ID No 10), Rv3868 (SEQID No 11), Rv3869 (SEQ ID No 12), Rv3870 (SEQ ID No 13), Rv3871 (SEQ IDNo 14), Rv3872 (SEQ ID No 15, mycobacterial PE), Rv3873 (SEQ ID No 16,PPE), Rv3874 (SEQ ID No 17, CFP-10), Rv3875 (SEQ ID No 18, ESAT-6),Rv3876 (SEQ ID No 19) and Rv3877 (SEQ ID No 20). In an another specificaspect, the invention relates to a strain of M. bovis BCG or M. microtiwherein said strain has integrated at least one, two, three or moregene(s) selected from Rv3871 (SEQ ID No 14), Rv3872 (SEQ ID No 15,mycobacterial PE), Rv3873(SEQ ID No 16,PPE), Rv3874(SEQ ID No17,CFP-10), Rv3875(SEQ ID No 18, ESAT-6) and Rv3876 (SEQ ID No 19).

Preferably, a strain according to the invention is one which hasintegrated a portion of DNA originating from Mycobacterium tuberculosisor any virulent member of the Mycobacterium tuberculosis complex (M.africanum, M. bovis, M. canettii), which comprises at least four genesselected from Rv3861 (SEQ ID No 4), Rv3862 (SEQ ID No 5), Rv3863 (SEQ IDNo 6), Rv3864 (SEQ ID No 7), Rv3865 (SEQ ID No 8), Rv3866 (SEQ ID No 9),Rv3867 (SEQ ID No 10), Rv3868 (SEQ ID No 11), Rv3869 (SEQ ID No 12),Rv3870 (SEQ ID No 13), Rv3871 (SEQ ID No 14), Rv3872 (SEQ ID No 15,mycobacterial PE), Rv3873 (SEQ ID No 16, PPE), Rv3814 (SEQ ID No 17,CFP-10), Rv3875 (SEQ ID No 18, ESAT-6), Rv3876 (SEQ ID No 19), Rv3877(SEQ ID No 20), Rv3878 (SEQ ID No 21), Rv3879 (SEQ ID No 22), Rv3880(SEQ ID No 23), Rv3881 (SEQ ID No 24), Rv3882 (SEQ ID No 25), Rv3883(SEQ ID No 26), Rv3884 (SEQ ID No 27) and Rv3885 (SEQ ID No 28),provided that it comprises Rv3874 (SEQ ID No 17, CFP-10) and/or Rv3875(SEQ ID No 18, ESAT-6).

Strains which have integrated a portion of DNA originating fromMycobacterium tuberculosis or any virulent member of the Mycobacteriumtuberculosis complex (M. Africanum, M. bovis, M. canettii)comprising atleast Rv3871 (SEQ ID No 14), Rv3875 (SEQ ID No 18, ESAT-6) and Rv3876(SEQ ID No 19) or at least Rv3871 (SEQ ID No 14), Rv3875 (SEQ ID No 18,ESAT-6) and Rv3877 (SEQ ID No 20) or at least Rv3871 (SEQ ID No 14),Rv3875 (SEQ ID No 18, ESAT-6), Rv3876 (SEQ ID No 19) and Rv3877 (SEQ IDNo 20) are of particular interest.

The above strains according to the invention may further comprise Rv3874(SEQ ID No 17, CFP-10), Rv3872 (SEQ ID No 15, mycobacterial PE) and/orRv3873 (SEQ ID No 16, PPE). In addition, it may further comprise atleast one, two, three or four gene(s) selected from Rv3861(SEQ ID No 4),Rv3862(SEQ ID No 5), Rv3863(SEQ ID No 6), Rv3864 (SEQ ID No 7), Rv3865(SEQ ID No 8), Rv3866 (SEQ ID No 9), Rv3867 (SEQ ID No 10), Rv3868 (SEQID No 11), Rv3869 (SEQ ID No 12), Rv3870 (SEQ ID No 13), Rv3878 (SEQ IDNo 21), Rv3879 (SEQ ID No 22), Rv3880 (SEQ ID No 23), Rv3881(SEQ IDNo.24), Rv3882(SEQ ID No25), Rv3883(SEQ ID No26), Rv3884 (SEQ ID No 27)and Rv3885 (SEQ ID No 28).

The invention encompasses strains which have integrated apportion of DNAoriginating from Mycobacterium tuberculosis or any virulent member ofthe Mycobacterium tuberculosis complex (M. africanum, M. bovis, M.canettii), which comprises Rv3875 (SEQ ID No 18, ESAT-6) or Rv3874 (SEQID No 17, CFP-10) or both Rv3875 (SEQ ID No 18, ESAT-6) and Rv3874 (SEQID No 17, CFP-10).

These genes can be mutated (deletion, insertion or base modification) soas to maintain the improved immunogenicity while decreasing thevirulence of the strains. Using routine procedure, the man skilled inthe art can select the M. bovis BCG::RD1 or M. microti::RD1 strains, inwhich a mutated gene has been integrated, showing improvedimmunogenicity and lower virulence.

We have shown here that introduction of the RD1-2F9 region makes thevaccine strains induce a more effective immune response against achallenge with M. tuberculosis. However, this first generation ofconstructs can be followed by other, more fine-tuned generations ofconstructs as the complemented BCG::RD1 vaccine strain also showed amore virulent phenotype in severely immuno-compromised (SCID) mice.Therefore, the BCG::RD1 constructs may be modified so as to beapplicable as vaccine strains while being safe for immuno-compromisedindividuals. The term “construct” means an engineered gene unit, usuallyinvolving a gene of interest that has been fused to a promoter.

In this perspective, the man skilled in the art can adapt the BCG::RD1strain by the conception of BCG vaccine strains that only carry parts ofthe genes coding for ESAT-6 or CFP-10 in a mycobacterial expressionvector (for example pSM81) tinder the control of a promoter, moreparticularly an hsp60 promoter. For example, at least one portion of theesat-6 gene that codes for immunogenic 20-mer peptides of ESAT-6 activeas T-cell epitopes (Mustafa AS, Oftung F, Amoudy HA, Madi NM, Abal AT,Shabant F, Rosen Krands I, & Andersen P. (2000) Multiple epitopes fromthe Mycobacterium tuberculosis ESAT-6 antigen are recognized byantigen-specific human T cell lines. Clin Infect Dis. 30 Suppl 3:S201-5,peptides P1 to P8 are incorporated herein in the description) could becloned into this vector and electroporated into BCG, resulting in a BCGstrain that produces these epitopes.

Alternatively, the ESAT-6 and CFP-10 encoding genes (for example onplasmid RD1-AP34 and or RD1-2F9) could be altered by directedmutagenesis (using for example QuikChange Site-Directed Mutagenesis Kitfrom Stratagen) in a way that most of the immunogenic peptides of ESAT-6remain intact, but the biological functionality of ESAT-6 is lost.

This approach could result in a more protective BCG vaccine withoutincreasing the virulence of the recombinant BCG strain.

Therefore, the invention is also aimed at a method for preparing andselecting M. bovis BCG or M. microti recombinant strains comprising astep consisting of modifying the M. bovis BCG::RD1 or M. microti::RD1strains as defined above by insertion, deletion or mutation in theintegrated RD1 region, more particularly in the esat-6 or CFP-10 gene,said method leading to strains that are less virulent forimmuno-depressed individuals. Together, these methods would allow toexplain what causes the effect that we see with our BCG::RD1 strain (thepresence of additional T-cell epitopes from ESAT-6 and CFP10 resultingin increased immunogenicity) or whether the effect is caused by betterfitness of the recombinant BCG::RD1 clones resulting in longer exposuretime of the immune system to the vaccine—or—by a combinatorial effect ofboth factors.

In a preferred embodiment, the invention is aimed at the M. bovisBCG::RD1 strains, which have integrated a cosmid herein referred to asthe RD1-2F9 and RD1-AP34 contained in the E. coli strains deposited onApr. 2, 2002 at the CNCM. (Institut Pasteur, 25, rue du Docteur Roux,75724 Paris cedex 15, France) under the accession number I-2831 andI-2832 respectively. The RDI-2F9 is a cosmid comprising the portion ofthe Mycobacterium tuberculosis H37Rv genome previously named RD1-2F9that spans the RD1 region and contains a gene conferring resistance toKanamycin. The RD1-AP34 is a cosmid comprising a portion of theMycobacterium tuberculosis H37Rv genome containing two genes coding forESAT-6 and CFP-10 as well as a gene conferring resistance to Kanamycin.

The cosmid RD1-AP34 contains a 3909 bp fragment of the M. tuberculosisH37Rv genome from region 4350459 bp to 4354367 bp that has been clonedinto an integrating vector pKint in order to be integrated in the genomeof Mycobacterium bovis BCG and Mycobacterium microti strains (SEQ ID No3). The Accession No. of the segment 160 of the M. tuberculosis H37Rvgenome that contains this region is AL022120. SEQ ID No 3:    1gaattcccat ccagtgagtt caaggtcaag cggcgccccc ctggccaggc atttctcgtc   61tcgccagacg gcaaagaggt catccaggcc ccctacatcg agcctccaga agaagtgttc  121gcagcacccc caagcgccgg ttaagattat ttcattgccg gtgtagcagg acccgagctc  181agcccggtaa tcgagttcgg gcaatgctga ccatcgggtt tgtttccggc tataaccgaa  241cggtttgtgt acgggataca aatacaggga gggaagaagt aggcaaatgg aaaaaatgtc  301acatgatccg atcgctgccg acattggcac gcaagtgagc gacaacgctc tgcacggcgt  361gacggccggc tcgacggcgc tgacgtcggt gaccgggctg gttcccgcgg gggccgatga  421ggtctccgcc caagcggcga cggcgttcac atcggagggc atccaattgc tggcttccaa  481tgcatcggcc caagaccagc tccaccgtgc gggcgaagcg gtccaggacg tcgcccgcac  541ctattcgcaa atcgacgacg gcgccgccgg cgtcttcgcc gaataggccc ccaacacatc  601ggagggagtg atcaccatgc tgtggcacgt aatgccaccg gagctaaata ccgcacggct  661gatggccggc gcgggtccgg ctccaatgct tgcggcggcc gcgggatggc agacgctttc  721ggcggctctg gacgctcagg ccgtcgagtt gaccgcgcgc ctgaactctc tgggagaagc  781ctggactgga ggtggcagcg acaaggcgct tgcggctgca acgccgatgg tggtctggct  841acaaaccgcg tcaacacagg ccaagacccg tgcgatgcag gcgacggcgc aagccgcggc  901atacacccag gccatggcca cgacgccgtc gctgccggag atcgccgcca accacatcac  961ccaggccgtc cttacggcca ccaacttctt cggtatcaac acgatcccga tcgcgttgac 1021cgagatggat tatttcatcc gtatgtggaa ccaggcagcc ctggcaatgg aggtctacca 1081ggccgagacc gcggttaaca cgcttttcga gaagctcgag ccgatggcgt cgatccttga 1141tcccggcgcg agccagagca cgacgaaccc gatcttcgga atgccctccc ctggcagctc 1201aacaccggtt ggccagttgc cgccggcggc tacccagacc ctcggccaac tgggtgagat 1261gagcggcccg atgcagcagc tgacccagcc gctgcagcag gtgacgtcgt tgttcagcca 1321ggtgggcggc accggcggcg gcaacccagc cgacgaggaa gccgcgcaga tgggcctgct 1381cggcaccagt acgctgtcga accatccgct ggctggtgga tcaggcccca gcgcgggcgc 1441gggcctgctg cgcgcggagt cgctacctgg cgcaggtggg tcgttgaccc gcacgccgct 1501gatgtctcag ctgatcgaaa agccggttgc cccctcggtg atgccggcgg ctgctgccgg 1561atcgtcggcg acgggtggcg ccgctccggt gggtgcggga gcgatgggcc agggtgcgca 1621atccggcggc tccaccaggc cgggtctggt cgcgccggca ccgctcgcgc aggagcgtga 1681agaagacgac gaggacgact gggacgaaga ggacgactgg tgagctcccg taatgacaac 1741agacttcccg gccacccggg ccggaagact tgccaacatt ttggcgagga aggtaaagag 1801agaaagtagt ccagcatggc agagatgaag accgatgccg ctaccctcgc gcaggaggca 1861ggtaatttcg agcggatctc cggcgacctg aaaacccaga tcgaccaggt ggagtcgacg 1921gcaggttcgt tgcagggcca gtggcgcggc gcggcgggga cggccgccca ggccgcggtg 1981gtgcgcttcc aagaagcagc caataagcag aagcaggaac tcgacgagat ctcgacgaat 2041attcgtcagg ccggcgtcca atactcgagg gccgacgagg agcagcagca ggcgctgtcc 2101tcgcaaatgg gcttctgacc cgctaatacg aaaagaaacg gagcaaaaac atgacagagc 2161agcagtggaa tttcgcgggt atcgaggccg cggcaagcgc aatccaggga aatgtcacgt 2221ccattcattc cctccttgac gaggggaagc agtccctgac caagctcgca gcggcctggg 2281gcggtagcgg ttcggaggcg taccagggtg tccagcaaaa atgggacgcc acggctaccg 2341agctgaacaa cgcgctgcag aacctggcgc ggacgatcag cgaagccggt caggcaatgg 2401cttcgaccga aggcaacgtc actgggatgt tcgca taggg caacgccgag ttcgcgtaga 2461atagcgaaac acgggatcgg gcgagttcga ccttccgtcg gtctcgccct ttctcgtgtt 2521tatacgtttg agcgcactct gagaggttgt catggcggcc gactacgaca agctcttccg 2581gccgcacgaa ggtatggaag ctccggacga tatggcagcg cagccgttct tcgaccccag 2641tgcttcgttt ccgccggcgc ccgcatcggc aaacctaccg aagcccaacg gccagactcc 2701gcccccgacg tccgacgacc tgtcggagcg gttcgtgtcg gccccgccgc cgccaccccc 2761acccccacct ccgcctccgc caactccgat gccgatcgcc gcaggagagc cgccctcgcc 2821ggaaccggcc gcatctaaac cacccacacc ccccatgccc atcgccggac ccgaaccggc 2881cccacccaaa ccacccacac cccccatgcc catcgccgga cccgaaccgg ccccacccaa 2941accacccaca cctccgatgc ccatcgccgg acctgcaccc accccaaccg aatcccagtt 3001ggcgcccccc agaccaccga caccacaaac gccaaccgga gcgccgcagc aaccggaatc 3061accggcgccc cacgtaccct cgcacgggcc acatcaaccc cggcgcaccg caccagcacc 3121gccctgggca aagatgccaa tcggcgaacc cccgcccgct ccgtccagac cgtctgcgtc 3181cccggccgaa ccaccgaccc ggcctgcccc ccaacactcc cgacgtgcgc gccggggtca 3241ccgctatcgc acagacaccg aacgaaacgt cgggaaggta gcaactggtc catccatcca 3301ggcgcggctg cgggcagagg aagcatccgg cgcgcagctc gcccccggaa cggagccctc 3361gccagcgccg ttgggccaac cgagatcgta tctggctccg cccacccgcc ccgcgccgac 3421agaacctccc cccagcccct cgccgcagcg caactccggt cggcgtgccg agcgacgcgt 3481ccaccccgat ttagccgccc aacatgccgc ggcgcaacct gattcaatta cggccgcaac 3541cactggcggt cgtcgccgca agcgtgcagc gccggatctc gacgcgacac agaaatcctt 3601aaggccggcg gccaaggggc cgaaggtgaa gaaggtgaag ccccagaaac cgaaggccac 3661gaagccgccc aaagtggtgt cgcagcgcgg ctggcgacat tgggtgcatg cgttgacgcg 3721aatcaacctg ggcctgtcac ccgacgagaa gtacgagctg gacctgcacg ctcgagtccg 3781ccgcaatccc cgcgggtcgt atcagatcgc cgtcgtcggt ctcaaaggtg gggctggcaa 3841aaccacgctg acagcagcgt tggggtcgac gttggctcag gtgcgggccg accggatcct 3901ggctctaga

pos. 0001-0006 EcoRI-restriction site

pos. 0286-0583 Rv3872 coding for a PE-Protein (SEQ ID No 15)

pos. 0616-1720 Rv3873 coding for a PPE-Protein (SEQ ID No 16)

pos. 1816-2115 Rv3874 coding for Culture Filtrat protein 10kD (CFP10)(SEQ ID No 17)

pos. 2151-2435 Rv3875 coding for Early Secreted Antigen Target 6kD(ESAT6) (SEQ ID No 18)

pos. 3903-3609 XbaI-restriction site

pos. 1816-2435 CFP-10 gene+esat-6 gene (SEQ ID No 29).

These sequences can be completed with the Rv3861 to Rv3871, and Rv3876to Rv3885 as referred in Table 1 below. Accesion number in NCBI Bank Loc(kb) in Coordinates in Molecular NC = gene M. Mycobacterium mass of GeneGene Protein Gene NP = tuberculosis tuberculosis protein Name lengthlength type protein H37Rv H37Rv (Dalton) Description Rv3861 324 108 CDS4337.95 4337946 . . . 11643.42 hypothetical 4338269 protein Rv3862 348116 CDS 4338.52 compl 12792.38 possible c-whiB6 4338174 . . .transcriptional 4338521 regulatory protein whiB- like WhiB6 Rv3863 1176392 CDS 4338.85 4338849 . . . 41087.44 hypothetical 4340024 alanine richprotein Rv3864 1206 402 CDS 4340.27 4340270 . . . 42068.66 conserved4341475 hypothetical protein Rv3865 309 103 CDS 4341.57 4341566 . . .10618.01 conserved 4341874 hypothetical protein Rv3866 849 283 CDS4341.88 4341880 . . . 30064.04 conserved 4342728 hypothetical proteinRv3867 549 183 CDS NC_000962 4342.77 4342767 . . . 19945.52 conservedNP_218384 4343318 protein Rv3868 1719 573 CDS NC_000962 4343.3 4343311 .. . 62425.40 conserved NP_218385 4345032 protein Rv3869 1440 480 CDSNC_000962 4345.04 4345036 . . . 51092.58 possible NP_218386 4346478conserved membrane protein Rv3870 2241 747 CDS NC_000962 4346.48 4346478. . . 80912.76 possible NP_218387 4348721 conserved membrane proteinRv3871 1773 591 CDS NC_000962 4348.83 4348824 . . . 64560.65 conservedNP_218388 4350599 protein Rv3876 1998 666 CDS NC_000962 4353.01 4353007. . . 70644.92 conserved NP_218393 4355007 proline and alanine richprotein Rv3877 1533 511 CDS NC_000962 4355.01 4355004 . . . 53981.12probable NP_218394 4356539 conserved transmembrane protein Rv3878 840280 CDS NC_000962 4356.69 4356693 . . . 27395.23 conserved 4357532hypothetical alanine rich protein Rv3879c 2187 729 CDS NC_000962 4359.78compl. 74492.13 hypothetical 4357596 . . . alanine and 4359782 prolinerich protein Rv3880c 345 115 CDS NC_000962 4360.55 compl. 12167.51conserved 4360202 . . . hypothetical 4360546 protein Rv3881c 1380 460CDS NC_000962 4361.92 compl. 47593.62 conserved 4360546 . . .hypothetical 4361925 alanine and glycine rich protein Rv3882c 1386 462CDS NC_000962 4363.42 compl. 50396.58 possible 4362035 . . . conservedmembrane 4363420 protein Rv3883c 1338 446 CDS NC_000962 4364.76 compl.45085.89 possible 4363420 . . . secreted 4364757 protease Rv3884c 1857619 CDS NC_000962 4366.84 compl. 68040.97 probable 4364982 . . .CBXX/CFQX 4366838 family protein Rv3885c 1611 537 CDS NC_000962 4368.52compl. 57637.95 possible 4366911 . . . conserved 4368521 membraneprotein

The sequence of the fragment RD1-2F9 (˜32 kb) covers the region of theM. tuberculosis genome AL123456 from ca 4337 kb to ca. 4369 kb, and alsocontains the sequence described in SEQ ID No 1. Therefore, the inventionalso embraces M. bovis BCG::RD1 strain and M. microti::RD1 strain whichhave integrated the sequence as shown in SEQ ID No 1.

The above described strains fulfill the aim of the invention which is toprovide an improved tuberculosis vaccine or M. bovis BCG-basedprophylactic or therapeutic agent, or a recombinant M. microtiderivative for these purposes.

The above described M. bovis BCG::RD1 strains are better tuberculosisvaccines than M. bovis BCG. These strains can also be improved byreintroducing other genes found in the RD8 and RD5 loci of M.tubercluosis or any virulent member of the Mycobacterium tuberculosiscomplex (M. africanum, M. bovis M. canettii). These regions code foradditional T-cell antigens.

As indicated, overexpressing the genes contained in the RD1, RD5 and RD8regions by means of exogenous promoters is encompassed by the invention.The same applies regarding M. microti::RD1 strains. M. microti strainscould also be improved by reintroducing the RD8 locus of M. tuberculosisor any virulent member of the Mycobacterium tuberculosis complex (M.africanum, M. bovis, M. canettii).

-    In a second embodiment, the invention is directed to a cosmid or a    plasmid, more commonly named vectors, comprising all or part of the    RD1-2F9 region originating from Mycobacterium tuberculosis or any    virulent member of the Mycobacterium tuberculosis complex (M.    africanum, M. bovis, M. canettii), said region comprising at least    ones two, three or more gene(s) selected from Rv3861 (SEQ ID No 4),    Rv3862 (SEQ ID No 5), Rv3863 (SEQ ID No 6), Rv3864 (SEQ ID No 7),    Rv3865 (SEQ ID No 8), Rv3866 (SEQ ID No 9), Rv3867 (SEQ ID No 10),    Rv3868 (SEQ ID No 11), Rv3869 (SEQ ID No 12), Rv3870 (SEQ ID No 13),    Rv3871 (SEQ ID No 14), Rv3872 (SEQ ID No 15, mycobacterial PE),    Rv3873 (SEQ ID No 16, PPE), Rv3874 (SEQ ID No 17, CFP-10), Rv3875    (SEQ ID No 18, ESAT-6), Rv3876 (SEQ ID No 19), Rv3877 (SEQ ID No    20), Rv3878 (SEQ ID No 21), Rv3879 (SEQ ID No 22), Rv3880 (SEQ ID No    23), Rv3881 (SEQ ID No 24), Rv3882 (SEQ ID No 25), Rv3883 (SEQ ID No    26), Rv3884 (SEQ ID No 27) and Rv3885 (SEQ ID No 28). The term    “vector” refers to a DNA molecule originating from a virus a    bacteria, or the cell of a higher organism into which another DNA    fragment of appropriate size can be integrated without loss of the    vectors capacity for self-replication; a vector introduces foreign    DNA into host cells, where it can be reproduced in large quantities.    Examples are plasmids, cosmids, and yeast artificial chromosomes;    vectors are often recombinant molecules containing DNA sequences    from several sources.

Preferably, a cosmid or a plasmid of the invention comprises a part ofthe RD1-2F9 region originating from Mycobacterium tuberculosis or anyvirulent member of the Mycobacterium tuberculosis complex (M. africanum,M. bovis, M. canettii), said part comprising at least one, two, three ormore gene(s) selected from Rv3867 (SEQ ID No 10), Rv3868 (SEQ ID No 11),Rv3869 (SEQ ID No 12), Rv3870 (SEQ ID No 13), Rv3871 (SEQ ID No 14),Rv3872 (SEQ ID No 15, mycobacterial PE), Rv3873 (SEQ ID No 16, PPE),Rv3874 (SEQ ID No 17, CFP-10), Rv3875 (SEQ ID No 18, ESAT6), Rv3876 (SEQID No 19) and Rv3877 (SEQ ID No 20).

Preferably, a cosmid or a plasmid of the invention comprises a part ofthe RD1-2F9 region originating from Mycobacterium tuberculosis or anyvirulent member of the Mycobacterium tuberculosis complex (M. africanum,M. bovis, M. canettii), said part comprising at least one, two, three ormore gene(s) selected from Rv3872 (SEQ ID No 15, mycobacterial PE),Rv3873 (SEQ ID No 16, PPE), Rv3874 (SEQ ID No 17, CFP-10), Rv3875 (SEQID No 18, ESAT-6) and Rv3876 (SEQ ID No 19).

Preferably, a cosmid or a plasmid of the invention comprises CFP-10,ESAT-6 or both or a part of them. It may also comprise a mutated geneselected CFP-10, ESAT-6 or both, said mutated gene being responsible forthe improved immunogenicity and decreased virulence.

A cosmid or a plasmid as mentioned above may comprise at least fourgenes selected from Rv3861 (SEQ ID No 4), Rv3862 (SEQ ID No 5), Rv3863(SEQ ID No 6), Rv3864 (SEQ ID No 7), Rv3865 (SEQ ID No 8), Rv3866 (SEQID No 9), Rv3867 (SEQ ID No 10), Rv3868 (SEQ ID No 11), Rv3869 (SEQ IDNo 12), Rv3870 (SEQ ID No 13), Rv3871 (SEQ ID No 14), Rv3872 (SEQ ID No15, mycobacterial PE), Rv3873 (SEQ ID No 16, PPE), Rv3874 (SEQ ID No 17,CFP-10), Rv3875 (SEQ ID No 18, ESAT-6), Rv3876 (SEQ ID No 19), Rv3877(SEQ ID No 20), Rv3878 (SEQ ID No 21), Rv3879 (SEQ ID No 22), Rv3880(SEQ ID No 23), Rv3881 (SEQ ID No 24), Rv3882 (SEQ ID No 25), Rv3883(SEQ ID No 26), Rv3884 (SEQ ID No 27) and Rv3885 (SEQ ID No 28),provided that it comprises Rv3874 (SEQ ID No 17, CFP-10) and/or Rv3875(SEQ ID No 18, ESAT-6)

Advantageously, a cosmid or a plasmid of the invention comprises aportion of DNA originating from Mycobacterium tuberculosis or anyvirulent member of the Mycobacterium tuberculosis complex (M. africanum,M. bovis, M. canettii), which comprises at least Rv3871 (SEQ ID No 14),Rv3875 (SEQ ID No 18, ESAT-6) and Rv3876 (SEQ ID No 19) or at leastRv3871 (SEQ ID No 14), Rv3875 (SEQ ID No 18, ESAT-6) and Rv3877 (SEQ IDNo 20) or at least Rv3871 (SEQ ID No 14), Rv3875 (SEQ ID No 18, ESAT-6),Rv3876 (SEQ ID No 19) and Rv3877 (SEQ ID No 20).

The above cosmids or plasmids may further comprise Rv3872 (SEQ ID No 15,mycobacterial PE) Rv3873 (SEQ ID No 16, PPE) Rv3874 (SEQ ID No 17,CFP-10). It may also further comprise at least one, two, three or fourgene(s) selected from Rv3861 (SEQ ID No 4), Rv3862 (SEQ ID No 5), Rv3863(SEQ ID No 6), Rv3864 (SEQ ID No 7), Rv3865 (SEQ ID No 8), Rv3866 (SEQID No 9), Rv3867 (SEQ ID No 10), Rv3868 (SEQ ID No 11), Rv3869 (SEQ IDNo 12), Rv3870 (SEQ ID No 13), Rv3878 (SEQ ID No 21), Rv3879 (SEQ ID No22), Rv3880 (SEQ ID No 23), Rv3881 (SEQ ID No 24), Rv3882 (SEQ ID No25), Rv3883 (SEQ ID No 26), Rv3884 (SEQ ID No 27) and Rv3885 (SEQ ID No28).

Two particular cosmids of the invention are the cosmids herein referredas RD1-2F9 and RD1-AP34 contained in the E. coli strains deposited atthe CNCM (Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris cedex15, France) under the accession number I-2831 and 1-2832 respectively.

A particular plasmid or cosmid of the invention is one which hasintegrated the complete RD1-2F9 region as shown in SEQ ID No 1.

The invention also relates to the use of these cosmids or plasmids fortransforming M. bovis BCG or M. microti strains.

As indicated above, these cosmids or plasmids may comprise a mutatedgene selected from Rv3861 to Rv3885, said mutated gene being responsiblefor the improved immunogenicity and decreased virulence.

In another embodiment, the invention embraces a pharmaceuticalcomposition comprising a strain as depicted above and a pharmaceuticallyacceptable carrier.

In addition to the strains, these pharmaceutical compositions maycontain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the livingvaccine into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Preferably, such composition is suitable for oral, intravenous orsubcutaneous administration.

The determination of the effective dose is well within the capability ofthose skilled in the art. A therapeutically effective dose refers tothat amount of active ingredient, i.e the number of strainsadministered, which ameliorates the symptoms or condition. Therapeuticefficacy and toxicity may be determined by standard pharmaceuticalprocedures in experimental animals, e.g., ED50 (the dose therapeuticallyeffective in 50% of the population) and LD50 (the dose lethal to 50% ofthe population). The dose ratio of toxic to therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. Of course, ED50 is to be modulated according to the mammal tobe treated or vaccinated. In this regard, the invention contemplates acomposition suitable for human administration as well as veterinarycomposition.

The invention is also aimed at a vaccine comprising a M. bovis BCG::RD1or M. microti::RD1 strain as depicted above and a suitable carrier. Thisvaccine is especially useful for preventing tuberculosis. It can also beused for treating bladder cancer.

The M. bovis BCG::RD1 or M. microti::RD1 strains are also useful as acarrier for the expression and presentation of foreign antigens ormolecules of interest that are of therapeutic or prophylactic interest.Owing to its greater persistence, BCG::RD1 will present antigens to theimmune system over a longer period thereby inducing stronger, morerobust immune responses and notably protective responses. Examples ofsuch foreign antigens can be found in patents and patent applicationsU.S. Pat. No. 6,191,270 for antigen LSA3, U.S. Pat. Nos. 6,096,879 and5,314,808 for HBV antigens, EP 201,540 for HIV-1 antigens, U.S. Pat. No.5,986,051 for H. pylori antigens and FR 2,744,724 for P. falciparumMSP-1 antigen.

The invention also concerns a product comprising a strain as depictedabove and at least one protein selected from ESAT-6 and CFP-10 orepitope derived thereof for a separate, simultaneous or sequential usefor treating tuberculosis.

In still another embodiment, the invention concerns the use of a M.bovis BCG::RD1 or M. microti::RD1 strain as depicted above forpreventing or treating tuberculosis. It also concerns the use of a M.bovis BCG::RD1 or M. microti::RD1 strain as a powerfuladjuvant/immunomodulator used in the treatment of superficial bladdercancer.

The invention also contemplates the identification at the species levelof members of the M. tuberculosis complex by means of an RD-basedmolecular diagnostic test. Inclusion of markers for RD1^(mic) andRD5^(mic) would improve the tests and act as predictors of virulence,especially in humans.

In this regard, the invention concerns a diagnostic kit for theidentification at the species level of members of the M. tuberculosiscomplex comprising DNA probes and primers specifically hybridizing to aDNA portion of the RD1 or RD5 region of M. tuberculosis, moreparticularly probes hybridizing under stringent conditions to a geneselected from Rv3871 (SEQ ID No 14), Rv3872 (SEQ ID No 15, mycobacterialPE), Rv3873 (SEQ ID No 16, PPE), Rv3874 (SEQ ID No 17, CFP-10), Rv3875(SEQ ID No 18, ESAT-6), and Rv3876 (SEQ ID No 19), preferably CFP-10 andESAT-6.

As used herein, the term “stringent conditions” refers to conditionswhich permit hybridization between the probe sequences and thepolynucleotide sequence to be detected. Suitably stringent conditionscan be defined by, for example, the concentrations of salt or formamidein the prehybridization and hybridization solutions, or by thehybridization temperature, and are well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature. The temperature range corresponding to a particular levelof stringency can be further narrowed by calculating the purine topyrimidine ratio of the nucleic acid of interest and adjusting thetemperature accordingly. Variations on the above ranges and conditionsare well known in the art.

Among the preferred primers, we can cite: primer esat-6FGTCACGTCCATTCATTCCCT, (SEQ ID No 32) primer esat-6RATCCCAGTGACGTTGCCTT), (SEQ ID No 33) primer RD1^(mic) flanking region FGCAGTGCAAAGGTGCAGATA, (SEQ ID No 34) primer RD1^(mic) flanking region RGATTGAGACACTTGCCACGA, (SEQ ID No 35) primer RD5^(mic) flanking region FGAATGCCGACGTCATATCG, (SEQ ID No 39) primer RD5^(mic) flanking region RCGGCCACTGAGTTCGATTAT. (SEQ ID No 40)

The present invention covers also the complementary nucleotide sequencesof said above primers as well as the nucleotide sequences hybridizingunder stringent conditions with them and having at least 20 nucleotidesand less than 500 nucleotides.

Diagnostic kits for the identification at the species level of membersof the M. tuberculosis complex comprising at least one, two, three ormore antibodies directed to mycobacterial PE, PPE, CFP-10, ESAT-6, arealso embraced by the invention.

Preferably, such kit comprises antibodies directed to CFP-10 and ESAT-6.

As used herein, the term “antibody” refers to intact molecules as wellas fragments thereof, such as Fab, F(ab').sub.2, and Fv, which arecapable of binding the epitopic determinant. Probes or antibodies can belabeled with isotopes, fluorescent or phosphorescent molecules or by anyother means known in the art.

The invention also relates to virulence markers associated with RD1and/or RD5 regions of the genome of M. tuberculosis or a part of theseregions.

The invention is further detailed below and will be illustrated with thefollowing figures.

FIGURE LEGENDS

FIG. 1: M. bovis BCG and M. microti have a chromosomal deletion, RD1,spanning the cfp10-esat6 locus.

(A) Map of the cfp10-esat6 region showing the six possible readingframes and the M. tuberculosis H37Rv gene predictions. This map is alsoavailable at: (http://genolist.pasteur.fr/TubercuList/).

The deleted regions are shown for BCG and M. microti with theirrespective H37Rv genome coordinates, and the extent of the conservedESAT-6 locus (F. Tekaia, et al., Tubercle Lung Disease 79, 329 (1999)),is indicated by the gray bar.

(B) Table showing characteristics of deleted regions selected forcomplementation analysis. Potential virulence factors and their putativefunctions disrupted by each deletion are shown. The coordinates are forthe M. tuberculosis H37Rv genome.

(C) Clones used to complement BCG. Individual clones spanning RD1regions (RD1-I106 and RD1-2F9) were selected from an ordered M.tuberculosis genomic library (R.B. unpublished) in pYUB412 (S. T. Cole,et al., Nature 393, 537 (1998) and W. R. Bange, F. M. Collins, W. R.Jacobs, Jr., Tuber. Lung Dis. 79, 171 (1999)) and electroporated into M.bovis BCG strains, or M. microti. Hygromycin-resistant transformantswere verified using PCR specific for the corresponding genes. pAP35 wasderived from RD1-2F9 by excision of an AflII fragment. pAP34 wasconstructed by subcloning an EcoRI-XbaI fragment into the integrativevector pKINT. The ends of each fragment are related to the BCG RD1deletion (shaded box) with black lines and the H37Rv coordinates for theother fragment ends given in kilobases.

(D)) Immunoblot analysis, using an ESAT-6 monoclonal antibody, of wholecell protein extracts from log-phase cultures of (well n°1) H37Rv (S. T.Cole, et al., Nature 393, 537 (1998)), (n°2) BCG::pYUB412 (M. A. Behr,et al., Science 284, 1520 (1999)), (n°3) BCG::RD1-I106 (R. Brosch, etal., Infection Immun. 66, 2221 (1998)), (n°4) BCG::RD1-2F9 (S. V.Gordon, et al., Molec Microbiol 32, 643 (1999)), (n°5) M. bovis (H.Salamon et al, Genome Res 10, 2044 (2000)), (n°6) Mycobacteriumsmegmatis (G. G. Mahairas, et al, J. Bacteriol. 178, 1274 (1996)), (n°7)M. smegmatis::pYUB412, and (n°8) M. smegmatis::RD1-2F9 (R. Brosch, etal., Proc Natl Acad Sci USA 99, 3684 (2002)).

FIG. 2: Complementation of BCG Pasteur with the RD1 region alters thecolony morphology and leads to accumulation of Rv3873 and ESAT-6 in thecell wall.

(A) Serial dilutions of 3 week old cultures of BCG::pYUB412, BCG::I106or BCG::RD1-2F9 growing on Middlebrook 7H10 agar plates. The whitesquare shows the area of the plate magnified in the image to the right.

(B) Light microscope image at fifty fold magnification of BCG::pYUB412and BCG::RD1-2F9 colonies. 5 μl drops of bacterial suspensions of eachstrain were spotted adjacently onto 7H10 plates and imaged after 10 daysgrowth, illuminating the colonies through the agar.

(C) Immunoblot analysis of different cell fractions of H37Rv obtainedfrom http://www.cvmbs.colostate.edu/microbioloy/tb/ResearchMA.html usingeither an anti-ESAT-6 antibody or

(D) anti-Rv3873 (PPE) rabbit serum. H37Rv and BCG signify whole cellextracts from the respective bacteria and Cyt, Mem and CW correspond tothe cytosolic, membrane and cell wall fractions of M. tuberculosisH37Rv.

FIG. 3: Complementation of BCG Pasteur with the RD1 region increasesbacterial persistence and pathogenicity in mice.

(A) Bacteria in the spleen and lungs of BALB/c mice followingintravenous (i.v.) infection via the lateral tail vein with 10⁶ colonyforming units (cfu) of M. tuberculosis H37Rv (black) or 10⁷ cfu ofeither BCG::pYUB412 (light grey) or BCG::RD1-I106 (grey).

(B) Bacterial persistence in the spleen and lungs of C57BL/6 micefollowing i.v. infection with 10⁵ cfu of BCG::pYUB412 (light grey),BCG::RD1-I106 (middle grey) or BCG::RD1-2F9 (dark grey).

(C) Bacterial multiplication after i.v. infection with 10⁶ cfu ofBCG::pYUB412 (light grey) and BCG::RD1-2F9 (grey) in severe combinedimmunodeficiency mice (SCID). For A, B, and C each timepoint is the meanof 3 to 4 mice and the error bars represent standard deviations.

(D) Spleens from SCID mice three weeks after i.v. infection with 10⁶ cfuof either BCG::pYUB412, BCG::RD1-2F9 or BCG::I301 (an RD3 “knock-in”,FIG. 1B). The scale is in cm.

FIG. 4: Immunisation of mice with BCG::RD1 generates marked ESAT-6specific T-cell responses and enhanced protection to a challenge with M.tuberculosis.

(A) Proliferative response of splenocytes of C57BL/6 mice immunisedsubcutaneously (s.c.) with 10⁶ CFU of BCG::pYUB412 (open squares) orBCG::RD1-2F9 (solid squares) to in vitro stimulation with variousconcentrations of synthetic peptides from poliovirus type 1 capsidprotein VP 1, ESAT-6 or Ag85A (K. Huygen, et al., Infect. Immun. 62, 363(1994), L. Brandt, J. Immunol. 157, 3527 (1996) and C. Leclerc et al, J.Virol. 65, 711 (1991)).

(B) Proliferation of splenocytes from BCG::RD1-2F9-immunised mice in theabsence or presence of 10 μg/ml of ESAT-6 1-20 peptide, with or without1 μg/ml of anti-CD4 (GK1.5) or anti-CD8 (H35-17-2) monoclonal antibody.Results are expressed as mean and standard deviation of ³H-thymidineincorporation from duplicate wells.

(C) Concentration of IFN-γ in culture supernatants of splenocytes ofC57BL/6 mice stimulated for 72 h with peptides or PPD after s.c. or i.v.immunisation with either BCG::pYUB412 (middle grey and white) orBCG::RD1-2F9 (light grey and black). Mice were inoculated with either10⁶ (white and light grey) or 10⁷ (middle grey and black) cfu. Levels ofIFN-γ were quantified using a sandwich ELISA (detection limit of 500pg/ml) with the mAbs R4-6A2 and biotin-conjugated XMG1.2. Results areexpressed as the mean and standard deviation of duplicate culture wells.

(D) Bacterial counts in the spleen and lungs of vaccinated andunvaccinated BALB/c mice 2 months after an i.v. challenge with M.tuberculosis H37Rv. The mice were challenged 2 months after i.v.inoculation with 10⁶ cfu of either BCG::pYUB412 or BCG::RD1-2F9. Organhomogenates for bacterial enumeration were plated on 7H11 medium, withor without hygromycin, to differentiate M. tuberculosis from residualBCG colonies. Results are expressed as the mean and standard deviationof 4 to 5 mice and the levels of significance derived using the Wilcoxonrang sum test.

FIG. 5: Mycobacterium microti strain OV254 BAC map (BAC clones namedMiXXX, where XXX is the identification number of the clone), overlaid onthe M. tuberculosis H37Rv (BAC clones named RvXXX, where XXX is theidentification number of the clone) and M. bovis AF2122/97 (BAC clonesnamed MbXXX, where XXX is the identification number of the clone) BACmaps. The scale bars indicate the position on the M. tuberculosisgenome.

FIG. 6: Difference in the region 4340-4360 kb between the deletion inBCG RD1^(beg) (A) and in M. microti RD1^(mic) (C) relative. to M.tuberculosis H37Rv (B).

FIG. 7: Difference in the region 3121-3127 kb between M. tuberculosisH37Rv (A) and M. microti OV254 (B). Gray boxes picture the directrepeats (DR), black ones the unique numbered spacer sequences. * spacersequence identical to the one of spacer 58 reported by van Embden et al.(42). Note that spacers 33-36 and 20-22 are not shown because H37Rvlacks these spacers.

FIG. 8: A) AseI PFGE profiles of various M. microti strains;Hybridization with a radiolabeled B) esat-6 probe; C) probe of theRD1^(mic) flanking region; D) plcA probe. 1. M. bovis AF2122/97, 2. M.canetti, 3. M. bovis BCG Pasteur, 4. M. tuberculosis H37Rv, 5. M.microti OV254, 6. M. microti Myc 94-2272, 7. M. microti B3 type mouse,8. M. microti B4 type mouse, 9. M. microti B2 type llama, 10. M. microtiB1 type llama, 11. M. microti ATCC 35782. M: Low range PFGE marker(NEB).

FIG. 9: PCR products obtained from various M. microti strains usingprimers that flank the RD1^(mic) region, for amplifying ESAT-6 antigen,that flank the MiD2 region. 1. M. microti B1 type llama, 2. M. microtiB4 type mouse, 3. M. microti B3 type mouse, 4. M. microti B2 type llama,5. M. microti ATCC 35782, 6. M. microti OV254, 7. M. microti Myc94-2272, 8. M. tuberculosis H37Rv.

FIG. 10: Map of the M. tuberculosis H37Rv RD1 genomic region. Map of thefragments used to complement BCG and M. microti (black) and the genomicregions deleted from different mycobacterial strains (grey). The middlepart shows key genes, putative promoters (P) and transcripts, thevarious proteins from the RD1 region, their sizes (number of amino acidresidues), InterPro domains (http://www.ebi.ac.uk/interpro/), membershipof M. tuberculosis protein families from TubercuList(http://genolist.pasteur.fr/TubercuList/). The dashed lines mark theextent of the RD1 deletion in BCG, M. microti and M. tuberculosisclinical isolate MT56 (Brosch, R, et al. A new evolutionary scenario forthe Mycobacterium tuberculosis complex. Proc Natl Acad Sci USA 99,3684-9. (2002)). M. bovis AF2122/97 is shown because it contains aframeshift mutation in Rv3881, a gene flanking the RD1 region of BCG.The fragments are drawn to show their ends in relation to the geneticmap, unless they extend beyond the genomic region indicated. pRD1-2F9,pRD1-I106 and pAP35 are based on pYUB412; pAP34 on pKINT; pAP47 andpAP48 on pSM81.

FIG. 11: Western blot analysis of various RD1 knock-ins of M. bovis BCGand M. microti. The left panel shows results of immunodetection ofESAT-6, CFP-10 and PPE68 (Rv3873) in whole cell lysates (WCL) andculture supernatants of BCG; the centre panel displays the equivalentfindings from M. microti and the right panel contains M. tuberculosisH37Rv control samples. Samples from mycobacteria transformed with thefollowing plasmids were present in lanes: -, pYUB412 vector control; 1,pAP34; 2, pAP35; 3, RD1-I106; 4, RD1-2F9. The positions of the nearestmolecular weight markers are indicated.

FIG. 12: Analysis of immune responses induced by BCG recombinants. A,The upper three panels display the results of splenocyte proliferationassays in response to stimulation in vitro with a peptide from MalE(negative control), to PPD or to a peptide containing an immunodominantCD4-epitope from ESAT-6. B, The lower panel shows IFN-γ production bysplenocytes in response to the same antigens. Symbols indicate thenature of the various BCG transformants. Samples were taken from C57BL/6mice immunised subcutaneously.

FIG. 13: Further immunological characterization of responses toBCG::RD1-2F9 A, Proliferative response of splenocytes of C57BL/6 miceimmunised subcutaneously (s.c.) with 10⁶ CFU of BCG::pYUB412 orBCG::RD1-2F9 to in vitro stimulation with various concentrations ofsynthetic peptides from poliovirus type 1 capsid protein VP1 (negativecontrol), ESAT-6 or Ag85A (see Methods for details). B, Proliferation ofsplenocytes from BCG::RD1-2F9-immunised mice in the absence or presenceof ESAT-6 1-20 peptide, with or without anti-CD4 or anti-CD8 monoclonalantibody. Results are expressed as mean and standard deviation of³H-thymidine incorporation from duplicate wells. c, Concentration ofIFN-γ in culture supernatants of splenocytes of C57BL/6 mice stimulatedfor 72 h with peptides or PPD after s.c. or i.v. immunisation witheither BCG::pYUB412 or BCG::RD1-2F9. Mice were inoculated with either10⁶ or 10⁷ CFU. Results are expressed as the mean and standard deviationof duplicate culture wells.

FIG. 14: Mouse protection studies. A, Bacterial counts in the spleen andlungs of vaccinated and unvaccinated C57BL/6 mice 2 months after an i.v.challenge with M. tuberculosis H37Rv. The mice were challenged 2 monthsafter i.v. inoculation with 10⁶ cfu of either BCG::pYUB412 orBCG::RD1-2F9. Organ homogenates for bacterial enumeration were plated on7H11 medium, with or without hygromycin, to differentiate M.tuberculosis from residual BCG colonies. Results are expressed as themean and standard deviation of 4 mice. Hatched columns correspond to thecohort of unvaccinated mice, while white and black columns correspond tomice vaccinated with BCG::pYUB412 and BCG::RD1-2F9, respectively. B,Bacterial counts in the spleen and lungs of vaccinated and unvaccinatedC57BL6 mice after an aerosol challenge with 1000 CFUs of M.tuberculosis. All mice were treated with antibiotics for three weeksprior to infection with M. tuberculosis. Data are the mean and SEmeasured on groups of three animals, and differences between groups wereanalysed using ANOVA (*p<0.05, **p<0.01).

FIG. 15: Guinea pig protection studies. A, Mean weight gain ofvaccinated and unvaccinated guinea pigs following aerosol infection withM. tuberculosis H37Rv. Guinea pigs were vaccinated with either saline(triangles), BCG (squares) or BCG::RD1-2F9 (filled circles). The errorbars are the standard error of the mean. Each time point represents themean weight of six guinea pigs. For the saline vaccinated group the lastlive weight was used for calculating the means as the animals werekilled on signs of severe tuberculosis which occurred after 50, 59, 71,72, 93 and 93 days. B, Mean bacterial counts in the spleen and lungs ofvaccinated and unvaccinated guinea pigs after an aerosol challenge withM. tuberculosis H37Rv. Groups of 6 guinea pigs were vaccinatedsubcutaneously with either saline, BCG or BCG::RD1-2F9 and infected 56days later. Vaccinated animals were killed 120 days following infectionand unvaccinated ones on signs of suffering or significant weight loss.The error bars represent the standard error of the mean of sixobservations. C, Spleens of vaccinated guinea pigs 120 days afterinfection with M. tuberculosis H37Rv; left, animal immunised with BCG;right, animal immunised with BCG::RD1-2F9.

FIG. 16: Diagram of the M. tuberculosis H37Rv genomic region showing aworking model for biogenesis and export. of ESAT-6 proteins. It presentsa possible functional model indicating predicted subcellularlocalization and potential interactions within the mycobacterial cellenvelope. Rosetta stone analysis indicates direct interaction betweenproteins Rv3870 and Rv3871, and the sequence similarity between theN-terminal domains of Rv3868 and Rv3876 suggests that these putativechaperones might also interact. Rv3868 is a member of the AAA-family ofATPases that perform chaperone-like functions by assisting in theassembly, and disassembly of protein complexes (Neuwald, A. F., Aravind,L., Spouge, J. L. & Koonin, E. V. AAA+: A class of chaperone-likeATPases associated with the assembly, operation, and disassembly ofprotein complexes. Genome Res 9, 27-43. (1999).). It is striking thatmany type III secretion systems require chaperones for stabilisation ofthe effector proteins that they secrete and for prevention of prematureprotein-protein interactions (Page, A. L. & Parsot, C. Chaperones of thetype III secretion pathway: jacks of all trades. Mol Microbiol 46, 1-11.(2002).). Thus, Rv3868, and possibly Rv3876,may be required for thefolding and/or dimerisation of ESAT-6/CFP-10 proteins (Renshaw, P. S.,et al. Conclusive evidence that the major T-cell antigens of the M.tuberculosis complex ESAT-6 and CFP-10 form a tight, 1:1 complex andcharacterisation of the structural properties of ESAT-6, CFP-10 and theESAT-6-CFP-10 complex: implications for pathogenesis and virulence. JBiol Chem 8, 8 (2002).), or even to prevent premature dimerisation.ESAT-6/CFP-10 are predicted to be exported through a transmembranechannel, consisting of at least Rv3870, Rv3871, and Rv3877,and possiblyRv3869, in a process catalysed by ATP-hydrolysis. Rv3873 (PPE 68) isknown to occur in the cell envelope and may also be involved as shownherein.

EXAMPLE 1 Preparation and Assessment of M. bovis BCG::RD1 Strains as aVaccine for Treating or Preventing Tuberculosis

As mentioned above, we have found that complementation with RD1 wasaccompanied by a change in colonial appearance as the BCG Pasteur“knock-in” strains developed a strikingly different morphotype (FIG.2A). The RD1 complemented strains adopted a spreading, less-rugosemorphology, that is characteristic of M. bovis, and this was moreapparent when the colonies were inspected by light microscopy (FIG. 2B).Maps of the clones used are shown (FIG. 1C). These changes were seenfollowing complementation with all of the RD1 constructs (FIG. 1C) andon complementing M. microti (data not shown). Pertinently, Calmette andGuérin (A. Calmette, La vaccination preventive contre la tuberculose.(Masson et cie., Paris, 1927)) observed a change in colony morphologyduring their initial passaging of M. bovis, and our experiments nowdemonstrate that this change, corresponding to loss of RD1, directlycontributed to attenuating this virulent strain. The integrity of thecell wall is known to be a key virulence determinant for M. tuberculosis(C. E. Barry, Trends Microbiol 9, 237 (2001)), and changes in both cellwall lipids (M. S. Glickman, J. S. Cox, W. R. Jacobs, Jr., Mol Cell 5,717 (2000)) and protein (F. X. Berthet, et al., Science 282, 759 (1998))have been shown to alter colony morphology and diminish persistence inanimal models.

To determine which genes were implicated in these morphological changes,antibodies recognising three RD1 proteins (Rv3873, CFP10 and ESAT-6)were used in immunocytological and subcellular fractionation analysis.When the different cell fractions from M. tuberculosis wereimmunoblotted all three proteins were localized in the cell wallfraction (FIG. 2C) though significant quantities of Rv3873, a PPEprotein, were also detected in the membrane and cytosolic fractions(FIG. 2D). Using immunogold staining and electron microscopy thepresence of ESAT-6 in the envelope of M. tuberculosis was confirmed butno alteration in capsular ultrastructure could be detected (data notshown). Previously, CFP-10 and ESAT-6 have been considered as secretedproteins (F. X. Berthet et al, Microbiology 144, 3195 (1998)) but ourresults suggest that their biological functions are linked directly withthe cell wall.

Changes in colonial morphology are often accompanied by alteredbacterial virulence. Initial assessment of the growth of differentBCG::RD1 “knock-ins” in C57BL/6 or BALB/c mice following intravenousinfection revealed that complementation did not restore levels ofvirulence to those of the reference strain M. tubercluosis H37Rv (FIG.3A). In longer-term experiments, modest yet significant differences weredetected in the persistence of the BCG::RD1 “knock-ins” in comparison toBCG controls. Following intravenous infection of C57BL/6 mice, only theRD1 “knock-ins” were still detectable in the lungs after 106 days (FIG.3B). This difference in virulence between the RD1 recombinants and theBCG vector control was more pronounced in severe combinedimmunodeficiency (SCID) mice (FIG. 3C). The BCG::RD1-2F9 “knock-in” wasmarkedly more virulent, as evidenced by the growth rate in lungs andspleen and also by an increased degree of splenomegaly (FIG. 3D).Cytological examination revealed numerous bacilli, extensive cellularinfiltration and granuloma formation. These increases in virulencefollowing complementation with the RD1 region, demonstrate that the lossof this genomic locus contributed to the attenuation of BCG.

The inability to restore full virulence to BCG Pasteur was not due toinstability of our constructs nor to the strain used (data not shown).Essentially identical results were obtained on complementing BCG Russia,a strain less passaged than BCG Pasteur and presumed, therefore, to becloser to the original ancestor (M. A. Behr, et al., Science 284, 1520(1999)). This indicates that the attenuation of BCG was a polymutationalprocess and loss of residual virulence for animals was documented in thelate 1920s (T. Oettinger, et al, Tuber Lung Dis 79, 243 (1999)). Usingthe same experimental strategy, we also tested the effects ofcomplementing with RD3-5, RD7 and RD9 (S. T. Cole, et al., Nature 393,537 (1998); M. A. Behr, et al., Science 284, 1520 (1999); R. Brosch, etal., Infection Immun. 66, 2221 (1998) and S. V. Gordon et al., MolecMicrobiol 32, 643 (1999)) encoding putative virulence factors (FIG. 1B).Reintroduction of these regions, which are not restricted to avirulentstrains, did not affect virulence in immuno-competent mice. Although itis possible that deletion effects act synergistically it seems moreplausible that other attenuating mechanisms are at play.

Since RD1 encodes at least two potent T-cell antigens (R. Colangelli, etal., Infect. Immun. 68, 990 (2000), M. Harboe, et al., Infect. Immun.66, 717 (1998) and R. L. V. SkjØt et al., Infect. Inmun. 68, 214(2000)), we investigated whether its restoration induced immuneresponses to these antigens or even improved the protective capacity ofBCG. Three weeks following either intravenous or subcutaneousinoculation with BCG::RD1 or BCG controls, we observed similarproliferation of splenocytes to an Ag85A (an antigenic BCG protein)peptide (K. Huygen, et al., Infect. Immun. 62, 363 (1994)), but notagainst a control viral peptide (FIG. 4A). Moreover, BCG::RD1 generatedpowerful CD4⁺T-cell responses against the ESAT-6 peptide as shown bysplenocyte proliferation (FIG. 4A, B) and strong IFN-γ production (FIG.4C). In contrast, the BCG::pYUB412 control did not stimulate ESAT-6specific T-cell responses thus indicating that these were mediated bythe RD1 locus. ESAT-6 is, therefore, highly immunogenic in mice in thecontext of recombinant BCG.

When used as a subunit vaccine, ESAT-6 elicits T-cell responses andinduces levels of protection weaker than but akin to those of BCG (L.Brandt et al, Infect. Immun. 68, 791 (2000)). Challenge experiments wereconducted to determine if induction of immune responses toBCG::RD1-encoded antigens, such as ESAT-6, could improve protectionagainst infection with M. tuberculosis. Groups of mice inoculated witheither BCG::pYUB412 or BCG::RD1 were subsequently infected intravenouslywith M. tuberculosis H37Rv. These experiments showed that immunisationwith the BCG::RD1 “knock-in” inhibited the growth of M. tuberculosiswithin both BALB/c (FIG. 4D) and C57BL/6 mice when compared toinoculation with BCG alone.

Although the increases in protection induced by BCG::RD1 and the BCGcontrol are modest they demonstrate convincingly that geneticdifferences have developed between the live vaccine and the pathogenwhich have weakened the protective capacity of BCG. This study thereforedefines the genetic basis of a compromise that has occurred, during theattenuation process, between loss of virulence and reduced protection(M. A. Behr, P. M. Small, Nature 389, 133 (1997)). The strategy ofreintroducing, or even overproducing (M. A. Horwitz et al, Proc NatlAcad Sci U S A 97, 13853 (2000)), the missing immunodominant antigens ofM. tuberculosis in BCG, could be combined with an immuno-neutralattenuating mutation to create a more efficacious tuberculosis vaccine.

EXAMPLE 2 BAC Based Comparative Genomics Identifies MycobacteriumMicroti as a Natural ESAT-6 Deletion Mutant

We searched for any genetic differences between human and vole isolatesthat might explain their different degree of virulence and hostpreference and what makes the vole isolates harmless for humans. In thisregard, comparative genomics methods were employed in connection withthe present invention to identify major differences that may existbetween the M. microti reference strain OV254 and the entirely sequencedstrains of M. tuberculosis H37Rv (10) or M. bovis AF2122/97 (14). Anordered Bacterial Artificial Chromosome (BAC) library of M. microtiOV254 was constructed and individual BAC to BAC comparison of a minimalset of these clones with BAC clones from previously constructedlibraries of M. tuberculosis H37Rv and M. bovis AF2122/97 wasundertaken.

Ten regions were detected in M. microti that were different to thecorresponding genomic regions in M. tuberculosis and M. bovis. Toinvestigate if these regions were associated with the ability of M.microti strains to infect humans, their genetic organization was studiedin 8 additional M. microti strains; including those isolated recentlyfrom patients with pulmonary tuberculosis. This analysis identified someregions that were specifically absent from all tested M. microtistrains, but present in all other members of the M. tuberculosis complexand other regions that were only absent from vole isolates of M.microti.

2.1 Material and Methods

Bacterial strains and plasmids. M. microti OV254 which was originallyisolated from voles in the UK in the 1930's was kindly supplied by M JColston (45). DNA from M. microti OV216 and OV183 were included in a setof strains used during a multicenter study (26). M. microti Myc 94-2272was isolated in 1988 from the perfusion fluid of a 41-year-old dialysispatient (43) and was kindly provided by L. M. Parsons. M. microti 35782was purchased from American Type Culture Collection (designation TMC1608 (M. P. Prague)). M. microti B1 type llama, B2 type llama, B3 typemouse and B4 type mouse were obtained from the collection of theNational Reference Center for Mycobacteria, Forschungszentrum Borstel,Germany. M. bovis strain AF2122/97, spoligotype 9 was responsible for aherd outbreak in Devon in the UK and has been isolated from lesions inboth cattle and badgers. Typically, mycobacteria were grown on 7H9Middlebrook liquid medium (Difco) containing 10%oleic-acid-dextrose-catalase (Difco), 0.2 % pyruvic acid and 0.05% Tween80.

Library construction, preparation of BAC DNA and sequencing reactions.Preparation of agarose-embedded genomic DNA from M. microti strainOV254, M. tuberculosis H37Rv, M. bovis BCG was performed as described byBrosch et al. (5). The M. microti library was constructed by ligation ofpartially digested HindIII fragments (50-125 kb) into pBeloBAC11. Fromthe 10,000 clones that were obtained, 2,000 were picked into 96 wellplates and stored at −80° C. Plasmid preparations of recombinant clonesfor sequencing reactions were obtained by pooling eight copies of 96well plates, with each well containing an overnight culture in 250 μl2YT medium with 12.5 μg.ml⁻¹ chloramphenicol. After 5 min centrifugationat 3000 rpm, the bacterial pellets were resuspended in 25 μl of solutionA (25 mM Tris, pH 8.0, 50 mM glucose and 10 mM EDTA), cells were lysedby adding 25 μl of buffer B (NaOH 0.2 M, SDS 0.2%). Then 20 μl of cold 3M sodium acetate pH 4.8 were added and kept on ice for 30 min. Aftercentrifugation at 3000 rpm for 30 min, the pooled supernatants (140 μl)were transferred to new plates. 130 μl of isopropanol were added, andafter 30 min on ice, DNA was pelleted by centrifugation at 3500 rpm for15 min. The supernatant was discarded and the pellet resuspended in 50μl of a 10 μg/ml RNAse A solution (in Tris 10 mM pH 7.5/EDTA 10 mM) andincubated at 64° C. for 15 min. After precipitation (2.5 μl of sodiumacetate 3 M pH 7 and 200 μl of absolute ethanol) pellets were rinsedwith 200 μl of 70% ethanol, air dried and finally suspended in 20 μl ofTE buffer.

End-sequencing reactions were performed with a Taq DyeDeoxy Terminatorcycle sequencing kit (Applied Biosystems) using a mixture of 13 μl ofDNA solution, 2 μl of Primer (2 μM) (SP6-BAC 1, AGTTAGCTCACTCATTAGGCA(SEQ ID No 15), or T7-BAC1, GGATGTGCTGCAAGGCGATTA (SEQ ID No 16)), 2.5μl of Big Dye and 2.5 μl of a 5×buffer (50 mM MgCl₂, 50 mM Tris).Thermal cycling was performed on a PTC-100 amplifier (MJ Inc.) with aninitial denaturation step of 60 s at 95° C., followed by 90 cycles of 15s at 95° C., 15 s at 56° C., 4 min at 60° C. DNA was then precipitatedwith 80 μl of 76% ethanol and centrifuged at 3000 rpm for 30 min. Afterdiscarding the supernatant, DNA was finally rinsed with 80 μl of 70%ethanol and resuspended in appropriate buffers depending on the type ofautomated sequencer used (ABI 377 or ABI 3700). Sequence data weretransferred to Digital workstations and edited using the TED softwarefrom the Staden package (37). Edited sequences were compared against theM. tuberculosis H37Rv database(http://genolist.pasteur.fr/TubercuList/), the M. bovis BLAST server(http://www.sanger.ac.uk/Projects/M. bovis/blast server.shtm1), andin-house databases to determine the relative positions of the M. microtiOV254 BAC end-sequences.

Preparation of BAC DNA from recombinants and BAC digestion profilecomparison. DNA for digestion was prepared as previously described (4).DNA (1 μg) was digested with HindIII (Boehringer) and restrictionproducts separated by pulsed-field gel electrophoresis (PFGE) on aBiorad CHEF-DR III system using a 1% (w/v) agarose gel and a pulse of3.5 s for 17 h at 6 V.cm⁻¹. Low-range PFGE markers (NEB) were used assize standards. Insert sizes were estimated after ethidium bromidestaining and visualization with UV light. Different comparisons weremade with overlapping clones from the M. microti OV254, M. bovisAF2122/97, and M. tuberculosis H37Rv pBeloBAC11 libraries.

PCR analysis to determine presence of genes in different M. microtistrains. Reactions contained 5 μl of 10×PCR buffer (100 mMβ-mercaptoethanol, 600 mM Tris-HCl, pH 8.8, 20 mM MgCl₂, 170 mM(NH₄)₂SO₄, 20 mM nucleotide mix dNTP), 2.5 μl of each primer at 2 μM, 10ng of template DNA, 10% DMSO and 0.5 unit pf Taq polymerase in a finalvolume of 12.5 μl. Thermal cycling was performed on a PTC-100 amplifier(MJ Inc.) with an initial denaturation step of 90 s at 95° C., followedby 35 cycles of 45 s at 95° C., 1 min at 60° C. and 2 min at 72° C.

RFLP analysis. In brief, agarose plugs of genomic DNA prepared aspreviously described (5) were digested with either AseI, DraI or XbaI(NEB), then electrophoresed on a 1% agarose gel, and finally transferredto Hybond-C extra nitrocellulose membranes (Amersham). Different probeswere amplified by PCR from the M. microti strain OV254 or M.tuberculosis H37Rv using primers for:

esat-6 (esat-6F GTCACGTCCATTCATTCCCT (SEQ ID No 17);

esat-6R ATCCCAGTGACGTTGCCTT) (SEQ ID No 18), the RD1^(mic) flankingregion (4340, 209F GCAGTGCAAAGGTGCAGATA (SEQ ID No 19); 4354,701RGATTGAGACACTTGCCACGA (SEQ ID No 20)), or plcA (plcA.int.FCAAGTTGGGTCTGGTCGAAT (SEQ ID No 21); plcA.int.R GCTACCCAAGGTCTCCTGGT(SEQ ID No 22)). Amplification products were radio-labeled by using theStratagene Prime-It II kit (Stratagene). Hybridizations were performedat 65° C. in a solution containing NaCl 0.8 M, EDTA pH 8, 5 mM, sodiumphosphate 50 mM pH 8, 2% SDS, 1× Denhardt's reagent and 100 μg/ml salmonsperm DNA (Genaxis). Membranes were exposed to phosphorimager screensand images were digitalized by using a STORM phospho-imager.

DNA sequence accession numbers. The nucleotide sequences that flankMiD1, MiD2, MiD3 as well as the junction sequence of RD1^(mic) have beendeposited in the EMBL database. Accession numbers are AJ345005,AJ345006, AJ315556 and AJ315557, respectively.

2.2 Results

Establishment of a complete ordered BAC library of M. microti OV254.Electroporation of pBeloBAC11 containing partial HindIII digests of M.microti OV254 DNA into Escherichia coli DH10B yielded about 10,000recombinant clones, from which 2,000 were isolated and stored in 96-wellplates. Using the complete sequence of the M. tuberculosis H37Rv genomeas a scaffold, end-sequencing of 384 randomly chosen M. microti BACclones allowed us to select enough clones to cover almost all of the 4.4Mb chromosome. A few rare clones that spanned regions that were notcovered by this approach were identified by PCR screening of pools aspreviously described (4). This resulted in a minimal set of 50 BACs,covering over 99.9% of the M. microti OV254 genome, whose positionsrelative to M. tuberculosis H37Rv are shown in FIG. 5. The insert sizeranged between 50 and 125 kb, and the recombinant clones were stable.Compared with other BAC libraries from tubercle bacilli (4, 13) the M.microti OV254 BAC library contained clones that were generally largerthan those obtained previously, which facilitated the comparativegenomics approach, described below.

Identification of DNA deletions in M. microti OV254 relative to M.tuberculosis H37Rv by comparative genomics. The minimal overlapping setof 50 BAC clones, together with the availability of three other orderedBAC libraries from M. tuberculosis H37Rv, M. bovis BCG Pasteur 1173P2(5, 13) and M. bovis AF2122/97 (14) allowed us to carry out direct BACto BAC comparison of clones spanning the same genomic regions. Sizedifferences of PFGE-separated HindIII restriction fragments from M.microti OV254 BACs, relative to restriction fragments from M. bovisand/or M. tuberculosis BAC clones, identified loci that differed amongthe tested strains. Size variations of at least 2 kb were easilydetectable and 10 deleted regions, evenly distributed around the genome,and containing more than 60 open reading frames (ORFs), were identified.These regions represent over 60 kb that are missing from M. microtiOV254 strain compared to M. tuberculosis H37Rv. First, it was found thatphiRv2 (RD11), one of the two M. tuberculosis H37Rv prophages waspresent in M. microti OV254, whereas phiRv1, also referred to as RD3(29) was absent. Second, it was found that M. microti lacks four of thegenomic regions that were also absent from M. bovis BCG. In fact, thesefour regions of difference named RD7, RD8, RD9 and RD10 are absent fromall members of the M. tuberculosis complex with the exception of M.tuberculosis and M. canettii, and seem to have been lost from a commonprogenitor strain of M. africanum, M. microti and M. bovis (3). As such,our results obtained with individual BAC to BAC comparisons show that M.microti is part of this non-M. tuberculosis lineage of the tuberclebacilli, and this assumption was further confirmed by sequencing thejunction regions of RD7-RD10 in M. microti OV254. The sequences obtainedwere identical to those from M. africanum, M. bovis and M. bovis BCGstrains. Apart from these four conserved regions of difference, andphiRv1 (RD3) M. microti OV254 did not show any other RDs with identicaljunction regions to M. bovis BCG Pasteur, which misses at least 17 RDsrelative to M. tuberculosis H37Rv (1, 13, 35). However, five otherregions missing from the genome of M. microti OV254 relative to M.tuberculosis H37Rv were identified (RD1^(mic), RD5^(mic), MiD1, MiD2,MiD3). Such regions are specific either for strain OV254 or for M.microti strains in general. Interestingly, two of these regions,RD1^(mic), RD5^(mic) partially overlap RDs from the M. bovis BCG.

Antigens ESAT-6 and CFP-10 are absent from M. microti. One of the mostinteresting findings of the BAC to BAC comparison was a novel deletionin a genomic region close to the origin of replication (FIG. 5).Detailed PCR and sequence analysis of this region in M. microti OV254showed a segment of 14 kb to be missing (equivalent to M. tuberculosisH37Rv from 4340,4 to 4354,5 kb) that partly overlapped RD1^(beg) absentfrom M. bovis BCG. More precisely, ORFs Rv3864 and Rv3876 are truncatedin M. microti OV254 and ORFs Rv3865 to Rv3875 are absent (FIG. 6). Thisobservation is particularly interesting as previous comparative genomicanalysis identified RD ₁ ^(beg) as the only RD region that isspecifically absent from all BCG sub-strains but present in all othermembers of the M. tuberculosis complex (1, 4, 13, 29, 35). As shown inFIG. 6, in M. microti OV254 the RD1^(mic) deletion is responsible forthe loss of a large portion of the conserved ESAT-6 family core region(40) including the genes coding for the major T-cell antigens ESAT-6 andCFP-10 (2, 15). The fact that previous deletion screening protocolsemployed primer sequences that were designed for the right hand portionof the RD1^(beg) region (i.e. gene Rv3878) (6, 39) explains why theRD1^(mic) deletion was not detected earlier by these investigations.FIG. 6 shows that RD1^(mic) does not affect genes Rv3877, Rv3878 andRv3879 which are part of the RD1^(beg) deletion.

Deletion of phospholipase-C genes in M. microti OV254. RD5^(mic), theother region absent from M. microti OV254, that partially overlapped anRD region from BCG, was revealed by comparison of BAC clone Mil8A5 withBAC Rv143 (FIG. 5). PCR analysis and sequencing of the junction regionrevealed that RD5^(mic) was smaller than the RD5 deletion in BCG (Table2 and 3 below). TABLE 2 Description of the putative function of thedeleted and truncated ORFs in M. microti OV254 Region Start-Endoverlapping ORF Putative Function or family RD 10 264,5-266,5Rv0221-Rv0223 echA1 RD 3 1779,5-1788,5 Rv1573-Rv1586 bacteriophageproteins RD 7 2207,5-2220,5 Rv1964-Rv1977 yrbE3A-3B; mce3A-F; unknown RD9 2330-2332 Rv2072-Rv2075 cobL; probable oxidoreductase; unknown RD5^(mic) 2627,6-2633,4 Rv2348-Rv2352 plc A-C; member of PPE family MiD13121,8-3126,6 Rv2816-Rv2819 IS6110 transposase; unknown MiD23554,0-3755,2 Rv3187-Rv3190 IS6110 transposase; unknown MiD33741,1-3755,7 Rv3345-Rv3349 members of the PE-PGRS and PPE families;insertion elements RD 8 4056,8-4062,7 Rv3617-Rv3618 ephA; lpqG; memberof the PE-PGRS family RD 1^(mic) 4340,4-4354,5 Rv3864-Rv3876 member ofthe CBXX/CF QX family; member of the PE and PPE families; ESAT-6; CFP10; unknown

TABLE 3 Sequence at the junction of the deleted regions in M. microtiOV254 Junction Position ORFs Sequences at the junction Flanking primersRD1^(mic) 4340, 421- Rv3864- CAAGACGAGGTTGTAAAACCTCGACG 4340, 209F (SEQID No 19) (SEQ ID 4354, 533 Rv3876 CAGGATCGGCGATGAAATGCCAGTCGGCAGTGCAAAGGTGCAGATA No 23) GCGTCGCTGAGCGCGCGCTGCGC CGA 4354, 701R (SEQID No 20) G TCCCATTTTGTCGCTGATTTGTTTGAACA GATTGAGACACTTGCCACGAGGGTCGGGGATTCCCT RD5^(mic) 2626, 831- Rv2349- CCTCGATGAACCACCTGACATGACCC2627, 370F (SEQ ID No 24) (SEQ ID 2635, 581 Rv2355CATCCTTTCCAAGAACTGGAGTCTCC GAATGCCGACGTCATATCG No 26)GGACATGCCGGGGCGGTTCA C TGCCC 2633, 692R (SEQ ID No 25)CAGGTGTCCTGGGTCGTTCCGTTGACCGT CGGCCACTGAGTTCGATTATCGAGTCCGAACATCCGTCATTCCCGGTGG CAGTCGGTGCGGTGAC MiD1 3121, 880- Rv2815c-CACCTGACATGACCCCATCCTTTCCA 3121, 690F (SEQ ID No 27) (SEQ ID 3126, 684Rv2818c AGAACTGGAGTCTCCGGACATGCCGG CAGCCAACACCAAGTAGACG No 29)GGCGGTTCAG GG ACATTCATGTCCATCTT 3126, 924R (SEQ ID No 28)CTGGCAGATCAGCAGATCGCTTGTTCTCAG TCTACCTGCAGTCGCTTGTG TGCAGGTGAGTC MiD23554, 066- Rv3188- GCTGCCTACTACGCTCAACGCCAGAG 3553, 880F (SEQ ID No 30)(SEQ ID 3555, 259 Rv3189 ACCAGCCGCCGGCTGAGGTCTCAGATGTCCATCGAGGATGTCTGAGT No 32) CAGAGAGTCTCCGGACTCACCGGGGC 3555, 385R (SEQID No 31) GGTTCA TAAAGGCTTCGAGACCGGACGG CTAGGCCATTCCGTTGTCTGGCTGTAGGTTCCTCAACTGTGTGGCGGAT GGTCTGAGCACTTAAC MiD3 3741, 139- Rv3345c-TGGCGCCGGCACCTCCGTTGCCACCG 3740, 950F (SEQ ID No 33) (SEQ ID 3755, 777Rv3349c TTGCCGCCGCTGGTGGGCGCGGTGCC GGCGACGCCATTTCC No 35)GTTCGCCCCGGCCCGAACCGTTCAGGG 3755, 988R (SEQ ID No 34) CCGGGTTCGCCCTCAGCCGCTAAACACG AACTGTCGGGCTTGCTCTT CCGACCAAGATCAACGAGCTACCTGCCCGGTCAAGGTTGAAGAGCCCCCATATCAGCA AGGGCCCGGTGTCGGCG

In fact, M. microti OV254 lacks the genes plcA, plcB, plcC and onespecific PPE-protein encoding gene (Rv2352). This was confirmed by theabsence of a clear band on a Southern blot of AseI digested genomic DNAfrom M. microti OV254 hybridized with a plcA probe. However, the genesRv2346c and Rv2347c, members of the esat-6 family, and Rv2348c, that aremissing from M. bovis and BCG strains (3) are still present in M.microti OV254. The presence of an IS6110 element in this segmentsuggests that recombination between two IS6110 elements could have beeninvolved in the loss of RD5^(mic), and this is supported by the findingthat the remaining copy of IS6110 does not show a 3 base-pair directrepeat in strain OV254 (Table 3).

Lack of MiD1 provides genomic clue for M. microti OV254 characteristicspoligotype. MiD1 encompasses the three ORFs Rv2816, Rv2817 and Rv2818that encode putative proteins whose functions are yet unknown, and hasoccurred in the direct repeat region (DR), a polymorphic locus in thegenomes of the tubercle bacilli that contains a cluster of directrepeats of 36 bp, separated by unique spacer sequences of 36 to 41 bp(17), (FIG. 7). The presence or absence of 43 unique spacer sequencesthat intercalate the DR sequences is the basis of spacer-oligo typing, apowerful typing method for strains from the M. tuberculosis complex(23). M. microti isolates exhibit a characteristic spoligotype with anunusually small DR cluster, due to the presence of only spacers 37 and38 (43). In M. microti OV254, the absence of spacers 1 to 36, which arepresent in many other M. tuberculosis complex strains, appears to resultfrom an IS6110 mediated deletion of 636 bp of the DR region.Amplification and PvuII restriction analysis of a 2.8 kb fragmentobtained with primers located in the genes that flank the DR region(Rv2813c and Rv2819) showed that there is only one copy of IS6110remaining in this region (FIG. 7). This IS6110 element is inserted intoORF Rv2819 at position 3,119,932 relative to the M. tuberculosis H37Rvgenome. As for other IS6110 elements that result from homologousrecombination between two copies (7), no 3 base-pair direct repeat wasfound for this copy of IS6110 in the DR region. Concerning the absenceof spacers 39-43 (FIG. 7), it was found that M. microti showed aslightly different organization of this locus than M. bovis strains,which also characteristically lack spacers 39-43. In M. microti OV254 anextra spacer of 36 bp was found that was not present in M. bovis nor inM. tuberculosis H37Rv. The sequence of this specific spacer wasidentical to that of spacer 58 reported by van Embden and colleagues(42). In their study of the DR region in many strains from the M.tuberculosis complex this spacer was only found in M. microti strainNLA000016240 (AF189828) and in some ancestral M. tuberculosis strains(3, 42). Like MiD1, MiD2 most probably results from an IS6110-mediateddeletion of two genes (Rv3188, Rv3189) that encode putative proteinswhose function is unknown (Table 3 above and Table 4 below). TABLE 4Presence of the RD and MiD regions in different M. microti strains HUMANB3 B4 B1 B2 HOST VOLES ATCC Myc 94- type type type type Strain OV 254 OV183 OV 216 35782 2272 mouse mouse llama llama RD1^(mic) absent absentabsent absent absent absent absent absent absent RD3 absent absentabsent absent absent absent absent absent absent RD7 absent absentabsent absent absent absent absent absent absent RD8 absent absentabsent absent absent absent absent absent absent RD9 absent absentabsent absent absent absent absent absent absent RD10 absent absentabsent absent absent absent absent absent absent MiD3 absent ND NDabsent absent absent absent absent absent MiD1 absent ND ND presentpartial partial partial present present RD5^(mic) absent absent absentpresent present present present present present MiD2 absent ND NDpresent present present present present presentND, not determined

Absence of some members of the PPE family in M. microti. MiD3 wasidentified by the absence of two HindIII sites in BAC Mi4B9 that existat positions 3749 kb and 3754 kb in the M. tuberculosis H37Rvchromosome. By PCR and sequence analysis, it was determined that MiD3corresponds to a 12 kb deletion that has truncated or removed five genesorthologous to Rv3345c-Rv3349c. Rv3347c encodes a protein of 3157amino-acids that belongs to the PPE family and Rv3346c a conservedprotein that is also present in M. leprae. The function of both theseputative proteins is unknown while Rv3348 and Rv3349 are part of aninsertion element (Table 2). At present, the consequences of the MiD3deletions for the biology of M. microti remains entirely unknown.

Extra-DNA in M. microti OV254 relative to M. tuberculosis H37Rv. M.microti OV254 possesses the 6 regions RvD1 to RvD5 and TBD1 that areabsent from the sequenced strain M. tuberculosis H37Rv, but which havebeen shown to be present in other members of the M. tuberculosiscomplex, like M. canettii, M. africanum, M. bovis, and M. bovis BCG (3,7, 13). In M. tuberculosis H37Rv, four of these regions (RvD2-5) containa copy of IS6110 which is not flanked by a direct repeat, suggestingthat recombination of two IS6110 elements was involved in the deletionof the intervening genomic regions (7). In consequence, it seemsplausible that these regions were deleted from the M. tuberculosis H37Rvgenome rather than specifically acquired by M. microti. In addition,three other small insertions have also been found and they are due tothe presence of an IS6110 element in a different location than in M.tuberculosis H37Rv and M. bovis AF2122/97. Indeed, PvuII RFLP analysisof M. microti OV254 reveals 13 IS6110 elements (data not shown).

Genomic diversity of M. microti strains. In order to obtain a moreglobal picture of the genetic organization of the taxon M. microti weevaluated the presence or absence of the variable regions found instrain OV254 in eight other M. microti strains. These strains which wereisolated from humans and voles have been designated as M. microti mainlyon the basis of their specific spoligotype (26, 32, 43) and can befurther divided into subgroups according to the host such as voles,llama and humans (Table 3). As stated in the introduction, M. microti israrely found in humans unlike M. tuberculosis. So the availability of 9strains from variable sources for genetic characterization is anexceptional resource. Among them was one strain (Myc 94-2272) from aseverely immuno-compromised individual (43), and four strains wereisolated from HIV-positive or HV-negative humans with spoligotypestypical of llama and mouse isolates. For one strain, ATCC 35872/M. P.Prague, we could not identify with certainty the original host fromwhich the strain was isolated, nor if this strain corresponds to M.microti OV166, that was received by Dr. Sula from Dr. Wells and usedthereafter for the vaccination program in Prague in the 1960's (38).

First, we were interested if these nine strains designated as M. microtion the basis of their spoligotypes also resembled each other by othermolecular typing criteria. As RFLP of pulsed-field gel separatedchromosomal DNA represents probably the most accurate molecular typingstrategy for bacterial isolates, we determined the Asel profiles of theavailable M. microti strains, and found that the profiles resembled eachother closely but differed significantly from the macro-restrictionpatterns of M. tuberculosis, M. bovis and M. bovis BCG strains used ascontrols. However, as depicted in FIG. 8A, the patterns were notidentical to each other and each M. microti strain showed subtledifferences, suggesting that they were not epidemiologically related. Asimilar observation was made with other rare cutting restrictionenzymes, like DraI or XbaI (data not shown).

Common and diverging features of M. microti strains. Two strategies wereused to test for the presence or absence of variable regions in thesestrains for which we do not have ordered BAC libraries. First, PCRsusing internal and flanking primers of the variable regions wereemployed and amplification products of the junction regions weresequenced. Second, probes from the internal portion of variable regionsabsent from M. microti OV254 were obtained by amplification of M.tuberculosis H37Rv DNA using specific primers. Hybridization with theseradio-labeled probes was carried out on blots from PFGE separated AseIrestriction digests of the M. microti strains. In addition, we confirmedthe findings obtained by these two techniques by using a focusedmacro-array, containing some of the genes identified in variable regionsof the tubercle bacilli to date (data not shown).

This led to the finding that the RD1^(mic) deletion is specific for allM. microti strains tested.

Indeed, none of the M. microti DNA-digests hybridized with theradio-labeled esat-6 probe (FIG. 8B) but with the RD1^(mic) flankingregion (FIG. 8C). In addition, PCR amplification using primers flankingthe RD1^(mic) region (Table 2) yielded fragments of the same size for M.microti strains whereas no products were obtained for M. tuberculosis,M. bovis and M. bovis BCG strains (FIG. 9). Furthermore, the sequence ofthe junction region was found identical among the strains which confirmsthat the genomic organization of the RD1^(mic) locus was the same in alltested M. microti strains (Table 3). This clearly demonstrates that M.microti lacks the conserved ESAT-6 family core region stretching inother members of the M. tuberculosis complex from Rv3864 to Rv3876 and,as such, represents a taxon of naturally occurring ESAT-6/CFP-10deletion mutants.

Like RD1^(mic), MiD3 was found to be absent from all nine M. microtistrains tested and, therefore, appears to be a specific genetic markerthat is restricted to M. microti strains (Table 3). However, PCRamplification showed that RD5^(mic) is absent only from the voleisolates OV254, OV216 and OV183, but present in the M. microti strainsisolated from human and other origins (Table 3). This was confirmed bythe presence of single bands but of differing sizes on a Southern blothybridized with a plcA probe for all M. microti tested strains exceptOV254 (FIG. 8D). Interestingly, the presence or absence of RD5^(mic)correlated with the similarity of IS6110 RFLP profiles. The profiles ofthe three M. microti strains isolated from voles in the UK differedconsiderably from the IS6110 RFLP patterns of humans isolates (43).Taken together, these results underline the proposed involvement ofIS6110 mediated deletion of the RD5 region and further suggest that RD5may be involved in the variable potential of M. microti strains to causedisease in humans. Similarly, it was found that MiD1 was missing onlyfrom the vole isolates OV254, OV216 and OV183, which display the samespoligotype (43), confirming the observations that MiD1 confers theparticular spoligotype of a group of M. microti strains isolated fromvoles. In contrast, PCR analysis revealed that MiD1 is only partiallydeleted from strains B3 and B4 both characterized by the mousespoligotype and the human isolate M. microti Myc 94-2272 (Table 3). Forstrain ATCC 35782 deletion of the MiD1 region was not observed. Thesefindings correlate with the described spoligotypes of the differentisolates, as strains that had intact or partially deleted MiD1 regionshad more spacers present than the vole isolates that only showed spacers37 and 38.

2.3 Comments and Discussion

We have searched for major genomic variations, due to insertion-deletionevents, between the vole pathogen, M. microti, and the human pathogen,M. tuberculosis. BAC based comparative genomics led to theidentification of 10 regions absent from the genome of the vole bacillusM. microti OV254 and several insertions due to IS6110. Seven of thesedeletion regions were also absent from eight other M. microti strains,isolated from voles or humans, and they account for more than 60 kb ofgenomic DNA.

Of these regions, RD1^(mic) is of particular interest, because absenceof part of this region has been found to be restricted to the BCGvaccine strains to date. As M. microti was originally described as nonpathogenic for humans, it is proposed here that RD1 genes is involved inthe pathogenicity for humans. This is reinforced by the fact thatRD1^(beg) (29) has lost putative ORFs belonging to the esat-6 genecluster including the genes encoding ESAT-6 and CFP-10 (FIG. 6) (40).Both polypeptides have been shown to act as potent stimulators of theimmune system and are antigens recognized during the early stages ofinfection (8, 12, 20, 34). Moreover, the biological importance of thisRD1 region for mycobacteria is underlined by the fact that it is alsoconserved in M. leprae, where genes ML0047-ML0056 show high similaritiesin their sequence and operon organization to the genes in the esat-6core region of the tubercle bacilli (11). In spite of the radical genedecay observed in M. leprae the esat-6 operon apparently has kept itsfunctionality in this organism.

However, the RD1 deletion may not be the only reason why the volebacillus is attenuated for humans. Indeed, it remains unclear whycertain M. microti strains included in the present study that showexactly the same RD1^(mic) deletion as vole isolates, have been found ascausative agents of human tuberculosis. As human M. microti cases areextremely rare, the most plausible explanation for this phenomenon wouldbe that the infected people were particularly susceptible formycobacterial infections in general. This could have been due to animmunodeficiency (32, 43) or to a rare genetic host predisposition suchas interferon gamma- or IL-12 receptor modification (22).

In addition, the finding that human M. microti isolates differed fromvole isolates by the presence of region RD5^(mic) may also have animpact on the increased potential of human M. microti isolates to causedisease. Intriguingly, BCG and the vole bacillus lack overlappingportions of this chromosomal region that encompasses three (plcA, plcB,plcC) of the four genes encoding phospholipase C (PLC) in M.tuberculosis. PLC has been recognized as an important virulence factorin numerous bacteria, including Clostridium perfringens, Listeriamonocytogenes and Pseudomonas aeruginosa, where it plays a role in cellto cell spread of bacteria, intracellular survival, and cytolysis (36,41). To date, the exact role of PLC for the tubercle bacilli remainsunclear. plA encodes the antigen mtp40 which has previously been shownto be absent from seven tested vole and hyrax isolates (28).Phospholipase C activity in M. tuberculosis, M. microti and M. bovis,but not in M. bovis BCG, has been reported (21, 47). However, PLC andsphingomyelinase activities have been found associated with the mostvirulent mycobacterial species (21). The levels of phospholipase Cactivity detected in M. bovis were much lower than those seen in M.tuberculosis consistent with the loss of plcABC. It is likely, that plcDis responsible for the residual phospholipase C activity in strainslacking RD5, such as M. bovis and M. microti OV254. Indeed, the plcDgene is located in region RvD2 which is present in some but not alltubercle bacilli (13, 18). Phospholipase encoding genes have beenrecognized as hotspots for integration of IS6110 and it appears that theregions RD5 and RvD2 undergo independent deletion processes morefrequently than any other genomic regions (44). Thus, the virulence ofsome M. microti strains may be due to a combination of functionalphospholipase C encoding genes (7, 25, 26, 29).

Another intriguing detail revealed by this study is that among thedeleted genes seven code for members of the PPE family of Gly-, Ala-,Asn-rich proteins. A closer look at the sequences of these genes showedthat in some cases they were small proteins with unique sequences, likefor example Rv3873, located in the RD1^(mic) region, or Rv2352c andRv2353c located in the RD5^(mic) region. Others, like Rv3347c, locatedin the MiD3 region code for a much larger PPE protein (3157 aa). In thiscase a neighboring gene (Rv3345c), belonging to another multigenefamily, the PE-PGRS family, was partly affected by the MiD3 deletion.While the function of the PE/PPE proteins is currently unknown, theirpredicted abundance in the proteome of M. tuberculosis suggests thatthey may play an important role in the life cycle of the tuberclebacilli. Indeed, recently some of them were shown to be involved in thepathogenicity of M. tuberculosis strains (9). Complementation of suchgenomic regions in M. microti OV254 should enable us to carry outproteomics and virulence studies in animals in order to understand therole of such ORFs in pathogenesis.

In conclusion, this study has shown that M. microti, a taxon originallynamed after its major host Microtus agrestis, the common vole,represents a relatively homogenous group of tubercle bacilli. Althoughall tested strains showed unique PFGE macro-restriction patterns thatdiffered slightly among each other, deletions that were common to all M.microti isolates (RD7-RD10, MiD3, RD1^(mic)) have been identified. Theconserved nature of these deletions suggests that these strains arederived from a common precursor that has lost these regions, and theirloss may account for some of the observed common phenotypic propertiesof M. microti, like the very slow growth on solid media and theformation of tiny colonies. This finding is consistent with results froma recent study that showed that M. microti strains carry a particularmutation in the gyrB gene (31).

Of particular interest, some of these common features (e.g. the flankingregions of RD1^(mic), or MiD3) could be exploited for an easy-to-performPCR identification test, similar to the one proposed for a range oftubercle bacilli (33). This test enables unambiguous and rapididentification of M. microti isolates in order to obtain a betterestimate of the overall rate of M. microti infections in humans andother mammalian species.

Example 3 Recombinant BCG Exporting ESAT-6 Confers Enhanced ProtectionAgainst Tuberculosis

3.1 Complementation of the RD1 locus of BCG Pasteur and M. microti

To construct a recombinant vaccine that secretes both ESAT-6 and CFP-10,we complemented BCG Pasteur for the RD1 region using genomic fragmentsspanning variable sections of the esxBA (or ESAT-6) locus from M.tuberculosis (FIG. 10). The RD1 deletion in BCG interrupts or removesnine CDS and affects all four transcriptional units: three are removedentirely while the fourth (Rv3867-Rv3871) is largely intact apart fromthe loss of 112 codons from the 3′-end of Rv3871 (FIG. 10).Transcriptome analysis of BCG, performed using cDNA probes obtained fromearly log phase cultures with oligonucleotide-based microarrays, wasable to detect signals at least two fold greater than background for theprobes corresponding to Rv3867 to 3871 inclusive, but not for theRD1-deleted genes Rv3872 to Rv3879. This suggests that the Rv3867-3871transcriptional unit is still active in BCG which, like M. bovis, alsohas frameshifts in the neighbouring gene, Rv3881 (FIG. 10). TheRD1^(mic) deletion of M. microti removes three transcriptional unitscompletely with only gene Rv3877 remaining from the fourth. The M.tuberculosis clinical isolate MT56 has lost genes Rv3878-Rv3879 (Brosch,R., et al. A new evolutionary scenario for the Mycobacteriumtuberculosis complex. Proc Natl Acad Sci U S A 99, 36849. (2002)) butstill secretes ESAT-6 and CFP-10 (FIG. 10).

To test the hypothesis that a dedicated export machinery exists and toestablish which genes were essential for creating an ESAT-6-CFP-10secreting vaccine we assembled a series of integrating vectors carryingfragments spanning different portions of the RD1 esx gene cluster (FIG.10). These integrating vectors stably insert into the attB site of thegenome of tubercle bacilli. pAP34 was designed to carry only theantigenic core region encoding ESAT-6 and CFP-10, and the upstream PEand PPE genes, whereas RD1-I106 and RD1-pAP35 were selected to includethe core region and either the downstream or upstream portion of thegene cluster, respectively. The fourth construct RD1-2F9 contains a ˜32kb segment from M. tuberculosis that stretches from Rv3861 to Rv3885covering the entire RD1 gene cluster. We adopted this strategy ofcomplementation with large genomic fragments to avoid polar effects thatmight be expected if a putative protein complex is only partiallycomplemented in trans. In addition, a set of smaller expressionconstructs (pAP47, pAP48) was established in which individual genes aretranscribed from a heat shock promoter (FIG. 10). Using appropriateantibodies all of these constructs were found to produce thecorresponding proteins after transformation of BCG or M. microti (seebelow).

3.2 Several genes of the esx cluster are required for export of ESAT-6and CFP-10

The four BCG::RD1 recombinants BCG::RD1-pAP34, BCG::RD1-pAP35,BCG::RD1-2F9 and BCG::RD1-I106) (FIG. 11) were initially tested toensure that ESAT-6 and CFP-10 were being appropriately expressed fromthe respective integrated constructs. Immunoblotting of whole cellprotein extracts from mid-log phase cultures of the various BCG::RD1recombinants using an ESAT-6 monoclonal antibody or polyclonal sera forCFP-10 and the PPE68 protein Rv3873 demonstrated that all three proteinswere expressed from the four constructs at levels comparable to those ofM. tuberculosis (FIG. 11). However, striking differences were seen whenthe supernatants from early log-phase cultures of each recombinant werescreened by Western blot for the two antigens. Although low levels ofESAT-6 and CFP-10 could be detected in the concentrated supernatantprotein fractions of BCG::RD1-pAP34, BCG::RD1-pAP35 and BCG::RD1-I106 itwas only with the integrated construct encompassing the entire esx genecluster (BCG::RD1-2F9) that the two antigens accumulated in significantamounts. The high concentrations of ESAT-6 and CFP-10 seen in thesupernatant of the recombinant BCG::RD1-2F9 were not due to anon-specific increase in permeability, or loss of cell wall material,because when the same whole cell and supernatant protein fractions wereimmunoblotted with serum raised against Rv3873, this protein was onlylocalized in the cell wall of the various recombinants. As expected,when constructs were used containing esxA or esxBA alone, ESAT-6 did notaccumulate in the culture supernatant (data not shown).

To assess the effect of the RD1^(mic) deletion of M. microti on theexport of ESAT-6 and CFP-10 and subsequent antigen handling, theexperiments were replicated in this genomic background. As with BCG,ESAT-6 and CFP-10 were only exported into the supernatant fraction insignificant amounts if expressed in conjunction with the entire esxcluster (FIG. 11). The combined findings demonstrate thatcomplementation with esxA or esxB alone is insufficient to produce arecombinant vaccine that secretes these two antigens. Rather, secretionrequires expression of genes located both upstream and downstream of theantigenic core region confirming our hypothesis ²⁰ that the conservedesx gene cluster does indeed encode functions essential for the exportof ESAT-6 and CFP-10.

3.3 Secretion of ESAT-6 is needed to induce antigen specific T-cellresponses

Since the classical observation that inoculation with live, but not deadBCG, confers protection against tuberculosis in animal models it hasbeen considered that secretion of antigens is critical for maximizingprotective T-cell immunity. Using our panel of recombinant vaccines wewere able to test if antigen secretion was indeed essential foreliciting ESAT-6 specific T-cell responses. Groups of C57/BL6 mice wereinoculated subcutaneously with one of six recombinant vaccines(BCG-pAP47, BCG-pAP48, BCG::RD1-pAP34, BCG::RD1-pAP35, BCG::RD1-I106,BCG::RD1-2F9) or with BCG transformed with the empty vector pYUB412.Three weeks following vaccination, T-cell immune responses to the sevenvaccines were assessed by comparing antigen-specific splenocyteproliferation and gamma interferon (IFN-γ) production (FIG. 12A). Asanticipated all of the vaccines generated splenocyte proliferation andIFN-γ production in response to PPD (partially purified proteinderivative) but not against an unrelated MalE control peptide indicatingsuccessful vaccination in each case. However, only splenocytes from themice inoculated with BCG::RD1-2F9 proliferated markedly in response tothe immunodominant ESAT-6 peptide (FIG. 12A). Furthermore, IFN-□ wasonly detected in culture supernatants of splenocytes from mice immunizedwith BCG::RD1-2F9 following incubation with the ESAT-6 peptide (FIG.12B) or recombinant CFP-10 protein (data not shown). These datademonstrate that export of the antigens is essential for stimulatingspecific Th1-oriented T-cells.

Further characterization of the immune responses was carried out.Splenocytes from mice immunized with BCG::RD1-2F9 or control BCG bothproliferated in response to the immunodominant antigen 85A peptide (FIG.13A). The strong splenocyte proliferation in the presence of ESAT-6 wasabolished by an anti-CD4 monoclonal antibody but not by anti-CD8indicating that the CD4⁺T-cell subset was involved (FIG. 13B).Interestingly, as judged by in vitro IFN-γ response to PPD and the ESATpeptide, subcutaneous immunization generated much stronger T-cellresponses (FIG. 13C) compared to intravenous injection. Aftersubcutaneous immunisation with BCG::RD1-2F9 strong ESAT-6 specificresponses were also detected in inguinal lymph nodes (data not shown).These experiments demonstrated that the ESAT-6 T-cell immune responsesto vaccination with BCG::RD1-2F9 were potent, reproducible and robustmaking this recombinant an excellent candidate for protection studies.

3.4 Protective efficacy of BCG::RD1-2F9 in immuno-competent mice

When used alone as a subunit or DNA vaccine, ESAT-6 induces levels ofprotection weaker than but akin to those of BCG (Brandt, L., Elhay, M.,Rosenkrands, I., Lindblad, E B. & Andersen, P. ESAT-6 subunitvaccination against Mycobacterium tuberculosis. Infect Immun. 68,791-795 (2000)). Thus, it was of interest to determine if thepresentation to the immune system of ESAT-6 and/or CFP-10 in the contextof recombinant BCG, mimicking the presentation of the antigens duringnatural infection, could increase the protective efficiency of BCG. TheBCG::RD1-2F9 recombinant was therefore selected for testing as avaccine, since it was the only ESAT-6 exporting BCG that elicitedvigorous antigen specific T-cell. immune responses. Groups of C57BL/6mice were inoculated intravenously with either BCG::RD1-2F9 orBCG::pYUB412 and challenged intravenously after eight weeks with M.tuberculosis H37Rv. Growth of M. tuberculosis H37Rv in spleens and lungsof each vaccinated cohort was compared with that of unvaccinatedcontrols two months after infection (FIG. 14A). This demonstrated that,compared to vaccination with BCG, the BCG::RD1-2F9 vaccine inhibitedgrowth of M. tuberculosis H37Rv in the spleens by 0.4 log10 CFU and wasof comparable efficacy at protecting the lungs.

To investigate this enhanced protective effect against tuberculosisfurther we repeated the challenge experiment using the aerosol route. Inthis experiment antibiotic treatment was employed to clear persistingBCG from mouse organs prior to infection with M. tuberculosis. Twomonths following vaccination C57BL/6 mice were treated with dailyrifampicin/izoniazid for three weeks and then infected with 1000 CFU ofM. tuberculosis H37Rv by the respiratory route. Mice were thensacrificed after 17, 35 and 63 days and bacterial enumeration carriedout on the lungs and spleen. This demonstrated that, even followingrespiratory infection, vaccination with BCG::RD1-2F9 was superior tovaccination with the control strain of BCG (FIG. 14B). However, growthof M. tuberculosis was again only inhibited strongly in the mousespleens.

Example 4 Protective Efficacy of BCG::RD1-2F9 in Guinea Pigs

4.1 Animal models M. tuberculosis H37Rv and the different recombinantvaccines were prepared in the same manner as for the immunologicalassays. For the guinea pig assays, groups of outbred femaleDunkin-Hartley guinea pigs (David Hall, UK) were inoculated with 5×10⁴CFUs by the subcutaneous route. Aerosol challenge was performed 8 weeksafter vaccination using a contained Henderson apparatus and an H37Rv(NCTC 7416) suspension in order to obtain an estimated retained inhaleddose of approximately 1000 CFU/lung (Williams, A., Davies, A, Marsh, P.D., Chambers, M. A & Hewinson, R. G. Comparison of the protectiveefficacy of bacille calmette-Guerin vaccination against aerosolchallenge with Mycobacterium tuberculosis and Mycobacterium bovis. ClinInfect Dis 30 Suppl 3, S299-301. (2000)). Organs were homogenized anddilutions plated out on 7H11 agar, as for the mice experiments. Guineapig experiments were carried out in the framework of the European UnionTB vaccine development program.

4.2 Results Although experiments in mice convincingly demonstrated asuperior protective efficacy of BCG::RD1 over BCG it was important toestablish a similar effect in the guinea pig model of tuberculosis.Guinea pigs are exquisitely sensitive to tuberculosis, succumbingrapidly to low dose infection with M. tuberculosis, and develop anecrotic granulomatous pathology closer to that of human tuberculosis.Immunization of guinea pigs with BCG::RD1-2F9 was therefore compared toconventional BCG vaccination. Groups of six guinea pigs were inoculatedsubcutaneously with saline, BCG or BCG::RD1-2F9. Eight weeks followinginoculation the three guinea pig cohorts were challenged with M.tuberculosis H37Rv via the aerosol route. Individual animals wereweighed weekly and were killed 17 weeks after challenge or earlier ifthey developed signs of severe tuberculosis. Whereas all unvaccinatedguinea pigs failed to thrive and were euthanised before the lasttime-point because of overwhelming disease, both the BCG- andrecombinant BCG::RD1-2F9-vaccinated animals progressively gained weightand were clinically well when killed on termination of the experiment(FIG. 15A). This indicated that although the BCG::RD1-2F9 recombinant ismore virulent in severely immunodeficient mice (Pym, A. S., Brodin, P.,

Brosch, R., Huerre, M. & Cole, S. T. Loss of RD1 contributed to theattenuation of the live tuberculosis vaccines Mycobacterium bovis BCGand Mycobacterium microti. Mol. Microbiol. 46, 709-717 (2002)). there isno increased pathogenesis in the highly susceptible guinea pig model oftuberculosis. Moreover, when the bacterial loads in the s spleens of thevaccinated animals were compared there was a greater than ten-foldreduction in the number of CFU recovered from the animals immunised withBCG::RD1-2F9 when compared to BCG (FIG. 15B). Interestingly, there wasno significant difference between the number of CFU obtained from thelungs of the two vaccinated groups indicating that the organ-specificenhanced protection observed in mice vaccinated with BCG::RD1-2F9 wasalso seen with guinea pigs. This marked reduction of bacterial loads inthe spleens of BCG::RD1-2F9 immunised animals was also reflected in thegross pathology. Visual examination of the spleens showed thattubercules were much larger and more numerous on the surface of theBCG-vaccinated guinea pigs (FIG. 15C). These results demonstrate thatthe recombinant vaccine BCG::RD1-2F9 conveys enhanced protection to anaerosol challenge with M. tuberculosis in two distinct animal models.

General Conclusion

Tuberculosis is still one of the leading infectious causes of death inthe world despite a decade of improving delivery of treatment andcontrol strategies (Dye, C., Scheele, S., Dolin, P., Pathania, V. &Raviglione, M. C. Consensus statement. Global burden of tuberculosis:estimated incidence, prevalence, and mortality by country. WHO GlobalSurveillance and Monitoring Project. Jama 282, 677-86. (1999)). Reasonsfor the recalcitrance of this pandemic are multi-factorial but includethe modest efficacy of the widely used vaccine, BCG. Two broadapproaches can be distinguished for the development of improvedtuberculosis vaccines (Baldwin,. S. L., et al. Evaluation of newvaccines in the mouse and guinea pig model of tuberculosis. Infection &Immunity 66, 2951-9 (1998), Kaufmann, S. H. How can immunologycontribute to the control of tuberculosis□ Nature Rev Immunol 1, 20-30.(2001) and Young, D. B. & Fruth, U. in New Generation Vaccines (eds.Levine, M., Woodrow, G., Kaper, J. & Cobon G S) 631-645 (Marcel Dekker,1997)). These are the development of subunit vaccines based on purifiedprotein antigens or new live vaccines that stimulate a broader range ofimmune responses. Although a growing list of individual or combinationsubunit vaccines, and hybrid proteins, have been tested none has yetproved superior to BCG in animal models (Baldwin, S. L., et al., 1998).Similarly, new attenuated vaccines derived from virulent M. tuberculosishave yet to out-perform BCG (Jackson, M., et a. Persistence andprotective efficacy of a Mycobacterium tuberculosis auxotroph vaccine.Infect Immun 67, 2867-73. (1999) and Hondalus, M. K., et al. Attenuationof and protection induced by a leucine auxotroph of Mycobacteniumtuberculosis. Infect Immun 68, 2888-98. (2000)). Interestingly, the onlyvaccine that appears to surpass BCG is a BCG recombinant over expressingantigen 85A (Horwitz, M. A., Harth, G., Dillon, B. J. & Maslesa-Galic,S. Recombinant bacillus calmette-guerin (BCG) vaccines expressing theMycobacterium tuberculosis 30-kDa major secretory protein induce greaterprotective immunity against tuberculosis than conventional BCG vaccinesin a highly susceptible animal model. Proc Natl Acad Sci U S A 97,i3853-8. (2000)). The basis for this vaccine was the notion thatover-expression of an immunodominant T-cell antigen could quantitativelyenhance the BCG-elicited immune response.

In frame with the invention, we were able to show that restoration ofthe RD1 locus did indeed improve the protective efficacy of BCG anddefines a genetic modification that should be included in newrecombinant BCG vaccines. Moreover, we were able to demonstrate twofurther findings that will be crucial for the development of a livevaccine against tuberculosis. First, we have identified the geneticbasis of secretion for the ESAT-6 family of immunodominant T-cellantigens, and second, we show that export of these antigens from thecytosol is essential for maximizing their antigenicity.

The extra-cellular proteins of M. tuberculosis have been extensivelystudied and shown to be a rich source of protective antigens (Sorensen,A. L., Nagai, S., Houen, G., Andersen, P. & Andersen, A. B. Purificationand characterization of a low-molecular-mass T-cell antigen secreted byMycobacterium tuberculosis. Infect Immun 63, 1710-7 (1995), SkjØt,R.L.V., et al. Comparative evaluation of low-molecular-mass proteinsfrom Mycobacterium tuberculosis identifies members of the ESAT-6 familyas immunodominant T-cell antigens. Infect. Immun 68, 214-220 (2000),Horwitz, M. A., Lee, B. W., Dillon, B. J. & Harth, G. Protectiveimmunity against tuberculosis induced by vaccination with majorextracellular proteins of Mycobacterium tuberculosis. Proc Natl Acad SciU S A 92, 1530-4 (1995) and Boesen, H., Jensen, B. N., Wilcke, T. &Andersen, P. Human T-cell responses to secreted antigen fractions ofMycobacterium tuberculosis. Infect Immun 63, 1491-7 (1995)). Despitethis it remains a mystery how some of these proteins, that lackconventional secretion signals, are exported from the cytosol, a uniqueproblem in M. tuberculosis given the impermeability and waxy nature ofthe mycobacterial cell envelope. Although two secA orthologues wereidentified in the genome sequence of M. tuberculosis (Cole, S. T., etal. Deciphering the biology of Mycobacterium tuberculosis from thecomplete genome sequence. Nature 393, 537-544 (1998)), no genes forobvious type I, II, or III protein secretion systems were detected, likethose that mediate the virulence of many Gram-negative bacterialpathogens (Finlay, B. B. & Falkow, S. Common themes in microbialpathogenicity revisited. Microbiol. Mol. Biol. Rev. 61, 136-169 (1997)).This suggested that novel secretion systems might exist. An in silhcoanalysis of the M. tuberculosis proteome identified a set of proteinsand genes whose inferred functions, genomic organisation and strictassociation with the esx gene family suggested that they couldconstitute such a system (Tekaia, F., et al. Analysis of the proteome ofMycobacterium tuberculosis in silico. Tubercle Lung Disease 79, 329-342(1999)). Our results provide the first empirical evidence that this genecluster is essential for the normal export of ESAT-6 and CFP-10.

The antigen genes, esxBA, lie at the centre of the conserved genecluster. Bioinformatics and comparative genomics predicted that both theconserved upstream genes Rv3868-Rv3871, as well as the downstream genesRv3876-Rv3877, would be required for secretion (FIG. 1) and strongexperimental support for this prediction is provided here. Ourexperiments show that only when BCG or M. microti are complemented withthe entire cluster is maximal export of ESAT-6 and CFP-10 obtained. Thissuggests that at least Rv3871 and either Rv3876 or Rv3877 are indeedessential for the normal secretion of ESAT-6 as these are the onlyconserved genes absent or disrupted in BCG which are not complemented byRD1-I106 or RD1-pAP35. These genes encode a large transmembrane proteinwith ATPase activity, an ATP-dependent chaperone and an integralmembrane protein, functional predictions compatible with them being partof a multi-protein complex involved in the translocation ofpolypeptides. Amongst the proteins encoded by the esx cluster Rv3871 andRv3877 are highly conserved, as orthologues have been identified in themore streamlined clusters found in other actinomycetes, furthersupporting their direct role in secretion (Gey Van Pittius, N. C., etal. The ESAT-6 gene cluster of Mycobacterium tuberculosis and other highG+C Gram-positive bacteria. Genome Biol 2, 44.1-44.18 (2001)). It hasbeen shown recently that ESAT-6 and CFP-10 form a heterodimer in vitro(Renshaw, P. S., et al. Conclusive evidence that the major T-cellantigens of the M. tuberculosis complex ESAT-6 and CFP-10 form a tight,1:1 complex and characterisation of the structural properties of ESAT-6,CFP-10 and the ESAT-6-CFP-10 complex: implications for pathogenesis andvirulence. J Biol Chem 8, 8 (2002)) but it is not known whetherdimerisation precedes translocation across the cell membrane or occursat a later stage in vivo. In either case, chaperone or protein clampactivity is likely to be required to assist dimer formation or toprevent premature complexes arising as is well documented for type IIIsecretion systems (Page, A. L. & Parsot, C. Chaperones of the type IIIsecretion pathway: jacks of all trades. Mol Microbiol 46, 1-11. (2002)).These, and other questions concerning the precise roles of theindividual components of the ESAT-6 secretory apparatus, can now beaddressed experimentally using the tools developed here.

The second major finding of the invention is that the secretion ofESAT-6 (and probably CFP-10) is critical for inducing maximal T-cellresponses although other RD1-encoded proteins may also contribute suchas the PPE68 protein (Rv3873) which is located in the cell envelope. Weshow that even though whole cell expression levels of ESAT-6 arecomparable amongst our vaccines (FIG. 2); only the vaccine strainexporting ESAT-6, via an intact secretory apparatus, elicits powerfulT-cell responses. Surprisingly, even the recombinants RD1-pAP47 andRD1-pAP48, that overexpress ESAT-6 intracellularly, did not generatedetectable ESAT-6 specific T-cell responses. Although antigen secretionhas long been recognized as important for inducing immunity against M.tuberculosis, and is often used to explain why killed BCG offers noprotection, this is one of the first fomial demonstrations of itsimportance. BCG, like M. tuberculosis resides in the phagosome, wheresecreted antigens have ready access to the MHC class II antigenprocessing pathway, essential for inducing IFN-γ producing CD4 T-cellsconsidered critical for protection against tuberculosis. Furtherunderstanding of the mechanism of ESAT-6 secretion could allow thedevelopment of BCG recombinants that deliver other antigens in the sameway.

The main aim of the present invention was to qualitatively enhance theantigenicity of BCG. So, having assembled a recombinant vaccine thatsecreted the T-cell antigens ESAT-6 and CFP-10, and shown that itelicited powerful CD4 T-cell immunity against at least ESAT-6 andCFP-10, the next step was to rigorously test its efficacy in animalmodels of tuberculosis. In three distinct models, including twoinvolving respiratory challenge, we were able to demonstrate that theESAT-6-CFP-10 secreting recombinant improved protection when compared toa BCG control, although this effect was restricted to the spleen. Thisis probably due to the fact that the enhanced immunity induced by thetwo additional antigens is insufficient to abort the primary infectionbut does significantly reduce the dissemination of bacteria from thelung. The lack of protection afforded to the lung, the portal of entryfor M. tuberculosis, does not prevent BCG::RD1-2F9 from being apromising vaccine candidate. Primary tuberculosis occurs in the middleand lower lobes and is rarely symptomatic (Garay, S. M. in Tuberculosis(eds. Rom, W. N. & Garay, S. M.) 373-413 (Little, Brown and Company,Boston, 1996)). The bacteria need to reach the upper lobes, thecommonest site of disease, by haematogenous spread. Therefore, a vaccinethat inhibits dissemination of M. tuberculosis from the primary site ofinfection would probably have major impact on the outcome oftuberculosis.

Recombinant BCG vaccines have definite advantages over other vaccinationstrategies in that they are inexpensive, easy to produce and convenientto store. However, despite an unrivalled and enviable safety recordconcerns remain and BCG is currently not administered to individualswith HIV infection. As shown above, the recombinant BCG::RD1-2F9 growsmore rapidly in Severe Combined Immunodeficient (SCID) mice, an extrememodel of immunodeficiency, than its parental BCG strain. However, inboth immunocompetent mice and guinea pigs we have not observed anyincreased pathology only a slight increase in persistence which may bebeneficial, since the declining efficacy of BCG with serial passage hasbeen attributed to an inadvertent increase in its attenuation (Behr, M.A. & Small, P. M. Has BCG attenuated to impotence□ Nature 389, 133-4.(1997)).

Ultimately, the robust enhancement in protection we have observed withthe reincorporation of the RD1 locus is a compelling reason to includethis genetic modification in any recombinant BCG vaccine, even if thismay require the need for a balancing attenuating mutation.

In summary, the data presented here show that, in addition to itsincreased persistence, BCG::RD1-2F9 induces specific T-cell memory andenhances immune responses to other endogenous Th1 antigens such as themycoloyl transferase, antigen 85A.

References

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1. A strain of M. bovis BCG or M. microti, wherein said strain hasintegrated all or part of the fragment, named RD1-2F9, of 31808 pb ofDNA originating from Mycobacterium tuberculosis or any virulent memberof the Mycobacterium tuberculosis complex (M. africanum, M. bovis, M.canettii), as shown in SEQ ID No 1 and which is responsible for enhancedimmunogenicity and increased persistence of BCG to the tubercle bacilli.2. A strain of M. bovis BCG or M. microti according to claim 1, whereinsaid strain has integrated all or part of the fragment of DNAoriginating from Mycobacterium tuberculosis or any virulent member ofthe Mycobacterium tuberculosis complex (M. africanum, M. bovis, M.canettii) as shown in SEQ ID No 2 responsible for enhancedimmunogenicity and increased persistence of BCG to the tubercle bacilli.3. A strain of M. bovis BCG or M. microti according to claim 1, whereinsaid strain has integrated all or part of the fragment of DNAoriginating from Mycobacterium tuberculosis or any virulent member ofthe Mycobacterium tuberculosis complex (M. africanum, M. bovis, M.canettii) as shown in SEQ ID No 3 responsible for enhancedimmunogenicity and increased persistence of BCG to the tubercle bacilli.4. A strain according to claim 1 which has integrated a portion of DNAoriginating from Mycobacterium tuberculosis or any virulent member ofthe Mycobacterium tuberculosis complex (M. africanum, M. bovis, M.canettii), which comprises at least one, two, three or more gene(s)selected from Rv3861 (SEQ ID No 4), Rv3862 (SEQ ID No 5), Rv3863 (SEQ IDNo 6), Rv3864 (SEQ ID No 7), Rv3865 (SEQ ID No 8), Rv3866 (SEQ ID No 9),Rv3867 (SEQ ID No 10), Rv3868 (SEQ ID No 11), Rv3869 (SEQ ID No 12),Rv3870 (SEQ ID No 13), Rv3871 (SEQ ID No 14), Rv3872 (SEQ ID No 15,mycobacterial PE), Rv3873 (SEQ ID No 16, PPE), Rv3874 (SEQ ID No 17,CFP-10), Rv3875 (SEQ ID No 18, ESAT-6), Rv3876 (SEQ ID No 19), Rv3877(SEQ ID No 20), Rv3878 (SEQ ID No 21), Rv3879 (SEQ ID No 22), Rv3880(SEQ ID No 23), Rv3881 (SEQ ID No 24), Rv3882 (SEQ ID No 25), Rv3883(SEQ ID No 26), Rv3884 (SEQ ID No 27) and Rv3885 (SEQ ID No 28).
 5. Astrain according to claim 1 which has integrated a portion of DNAoriginating from Mycobacterium tuberculosis or any virulent member ofthe Mycobacterium tuberculosis complex (M. africanum, M. bovis, M.canettii), which comprises at least one, two, three or more gene(s)selected from Rv3867 (SEQ ID No 10), Rv3868 (SEQ ID No 11), Rv3869 (SEQID No 12), Rv3870 (SEQ ID No 13), Rv3871 (SEQ ID No 14), Rv3872 (SEQ IDNo 15, mycobacterial PE), Rv3873 (SEQ ID No 16, PPE), Rv3874 (SEQ ID No17, CFP-10), Rv3875 (SEQ ID No 18, ESAT-6), Rv3876 (SEQ ID No 19) andRv3877 (SEQ ID No 20).
 6. A strain according to claim 1 which hasintegrated a portion of DNA originating from Mycobacterium tuberculosisor any virulent member of the Mycobacterium tuberculosis complex (M.africanum, M. bovis, M. canettii), which comprises at least one, two,three or more gene(s) selected from Rv3872 (SEQ ID No 15, mycobacterialPE), Rv3873 (SEQ ID No 16,PPE), Rv3874 (SEQ ID No 17, CFP-10) and Rv3875(SEQ ID No 18, ESAT-6).
 7. A strain according to claim 1 which hasintegrated a portion of DNA originating from Mycobacterium tuberculosisor any virulent member of the Mycobacterium tuberculosis complex (M.africanum, M. bovis, M. canettii), which comprises at least four genesselected from Rv3861 (SEQ ID No 4), Rv3862 (SEQ ID No 5), Rv3863 (SEQ IDNo 6), Rv3864 (SEQ ID No 7), Rv3865 (SEQ ID No 8), Rv3866 (SEQ ID No 9),Rv3867 (SEQ ID No 10), Rv3868 (SEQ ID No 11), Rv3869 (SEQ ID No 12),Rv3870 (SEQ ID No 13), Rv3871 (SEQ ID No 14), Rv3872 (SEQ ID No 15,mycobacterial PE), Rv3873 (SEQ ID No 16, PPE), Rv3874 (SEQ ID No 17,CFP-10), Rv3875 (SEQ ID No 18, ESAT-6), Rv3876 (SEQ ID No 19), Rv3877(SEQ ID No 20), Rv3878 (SEQ ID No 21), Rv3879 (SEQ ID No 22), Rv3880(SEQ ID No 23), Rv3881 (SEQ ID No 24), Rv3882 (SEQ ID No 25), Rv3883(SEQ ID No 26), Rv3884 (SEQ ID No 27) and Rv3885 (SEQ ID No 28),provided that it comprises Rv3874 (SEQ ID No 17, CFP-10) and/or Rv3875(SEQ ID No 18, ESAT-6).
 8. A strain according to claim 1 which hasintegrated a portion of DNA originating from Mycobacterium tuberculosisor any virulent member of the Mycobacterium tuberculosis complex (M.africanum, M. bovis, M. canettii), which comprises at least Rv3871 (SEQID No 14), Rv3875 (SEQ ID No 18, ESAT-6) and Rv3876 (SEQ ID No 19).
 9. Astrain according to claim 1 which has integrated a portion of DNAoriginating from Mycobacterium tuberculosis or any virulent member ofthe Mycobacterium tuberculosis complex (M. africanum, M. bovis, M.canettii), which comprises at least Rv3871 (SEQ ID No 14), Rv3875 (SEQID No 18, ESAT-6) and Rv3877 (SEQ ID No 20).
 10. A strain according toclaim 1 which has integrated a portion of DNA originating fromMycobacterium tuberculosis or any virulent member of the Mycobacteriumtuberculosis complex (M. africanum, M. bovis, M. canettii), whichcomprises at least Rv3871 (SEQ ID No 14), Rv3875 (SEQ ID No 18, ESAT-6),Rv3876 (SEQ ID No 19) and Rv3877 (SEQ ID No 20).
 11. A strain accordingto one of claims 8 to 10 which has integrated a portion of DNAoriginating from Mycobacterium tuberculosis or any virulent member ofthe Mycobacterium tuberculosis complex (M. africanum, M. bovis, M.canettii), which further comprises Rv3874 (SEQ ID No 17, CFP-10).
 12. Astrain according to one of claims 8 to 11 which has integrated a portionof DNA originating from Mycobacterium tuberculosis or any virulentmember of the Mycobacterium tuberculosis complex (M. africanum, M.bovis, M. canettii), which further comprises Rv3872 (SEQ ID No 15,mycobacterial PE).
 13. A strain according to one of claims 8 to 12 whichhas integrated a portion of DNA originating from Mycobacteriumtuberculosis or any virulent member of the Mycobacterium tuberculosiscomplex (M. africanum, M. bovis, M. canettii), which further comprisesRv3873 (SEQ ID No 16, PPE).
 14. A strain according to one of claims 8 to13 which has integrated a portion of DNA originating from Mycobacteriumtuberculosis or any virulent member of the Mycobacterium tuberculosiscomplex (M. africanum, M. bovis, M. canettii), which further comprisesat least one, two, three or four gene(s) selected from Rv3861 (SEQ ID No4), Rv3862 (SEQ ID No 5), Rv3863 (SEQ ID No 6), Rv3864 (SEQ ID No 7),Rv3865 (SEQ ID No 8), Rv3866 (SEQ ID No 9), Rv3867 (SEQ ID No 10),Rv3868 (SEQ ID No 11), Rv3869 (SEQ ID No 12), Rv3870 (SEQ ID No 13),Rv3878 (SEQ ID No 21), Rv3879 (SEQ ID No 22), Rv3880 (SEQ ID No 23),Rv3881 (SEQ ID No 24), Rv3882 (SEQ ID No 25), Rv3883 (SEQ ID No 26),Rv3884 (SEQ ID No 27) and Rv3885 (SEQ ID No 28).
 15. A strain accordingto claim 1 which has integrated a portion of DNA originating fromMycobacterium tuberculosis or any virulent member of the Mycobacteriumtuberculosis complex (M. africanum, M. bovis, M. canettii), whichcomprises Rv3875 (SEQ ID No 18, ESAT-6).
 16. A strain according to claim1 which has integrated a portion of DNA originating from Mycobacteriumtuberculosis or any virulent member of the Mycobacterium tuberculosiscomplex (M. africanum, M. bovis, M. canettii), which comprises Rv3874(SEQ ID No 17, CFP-10).
 17. A strain according to claim 1 which hasintegrated a portion of DNA originating from Mycobacterium tuberculosisor any virulent member of the Mycobacterium tuberculosis complex (M.africanum, M. bovis, M. canettii), which comprises both Rv3875 (SEQ IDNo 18, ESAT-6) and Rv3874 (SEQ ID No 17, CFP-10).
 18. A strain accordingto one of claims 4 to 17, wherein the coding sequence of the integratedgene is in frame with its natural promoter or with an exogenouspromoter, such as a promoter capable of directing high level ofexpression of said coding sequence.
 19. A strain according to one ofclaims 4 to 17, wherein the said integrated gene is mutated so as tomaintain the improved immunogenicity while decreasing the virulence ofthe strain.
 20. A strain according to claim 18 or 19, wherein saidstrain only carries parts of the genes coding for ESAT-6 or CFP-10 in amycobacterial expression vector under the control of a promoter, moreparticularly an hsp60 promoter.
 21. A strain according to claim 18,wherein said strain carries at least one portion of the esat-6 gene thatcodes for immunogenic 20-mer peptides of ESAT-6 active as T-cellepitopes.
 22. A strain according to claim 19, wherein the esat-6encoding gene is altered by directed mutagenesis in a way that most ofthe immunogenic peptides of ESAT-6 remain intact, but the biologicalfunctionality of ESAT-6 is lost.
 23. A strain according to claim 19,wherein the CFP-10 encoding gene is altered by directed mutagenesis in away that most of the immunogenic peptides of CFP-10 remain intact, butthe biological functionality of CFP-10 is lost.
 24. M. bovis BCG::RD1strains which have integrated a cosmid herein referred to as RD1-2F9 andRD1-AP34 contained in the E. coli strains deposited at the CNCM underthe accession number I-2831 and I-2832 respectively.
 25. M. bovisBCG::RD1 strain which has integrated the insert of the cosmid RD1-AP34which corresponds to the 3909 bp fragment of the M. tuberculosis H37Rvgenome from region 4350459 bp to 4354367 bp cloned as shown in SEQ ID No3.
 26. M. bovis BCG::RD1 strain which has integrated the insert of thecosmid RD1-2F9 (˜32 kb) that covers the region of the M. tuberculosisgenome AL123456 from ca 4337 kb to ca. 4369 kb as shown in SEQ ID No 1.27. M. microti::RD1 strain which has integrated the insert of the cosmidRD1-AP34 which corresponds to the 3909 bp fragment of the M.tuberculosis H37Rv genome from region 4350459 bp to 4354367 bp cloned asshown in SEQ ID No 3).
 28. M. microti::RD1 strain which has integratedthe insert of the cosmid RD1 -2F9 (˜32 kb) that covers the region of theM. tuberculosis genome AL123456 from ca 4337 kb to ca. 4369 kb as shownin SEQ ID No
 1. 29. A method for preparing and selecting improved M.bovis BCG or M. microti strains defined in any one of claims 1 to 28comprising a step consisting of modifying said strains by insertion,deletion or mutation in the integrated portion of DNA originating fromMycobacterium tuberculosis or any virulent member of the Mycobacteriumtuberculosis complex (M. africanum, M. bovis, M. canettii), moreparticularly in the esat-6 or CFP-10 gene, said method leading tostrains that are less virulent for immuno-depressed individuals.
 30. Acosmid or a plasmid comprising a portion of DNA originating fromMycobacterium tuberculosis or any virulent member of the Mycobacteriumtuberculosis complex (M. africanum, M. bovis, M. canettii), said portionof DNA comprising at least one, two, three or more gene(s) selected fromRv3861 (SEQ ID No 4), Rv3862 (SEQ ID No 5), Rv3863 (SEQ ID No 6), Rv3864(SEQ ID No 7), Rv3865 (SEQ ID No 8), Rv3866 (SEQ ID No 9), Rv3867 (SEQID No 10), Rv3868 (SEQ ID No 11), Rv3869 (SEQ ID No 12), Rv3870 (SEQ IDNo 13), Rv3871 (SEQ ID No 14), Rv3872 (SEQ ID No 15, mycobacterial PE),Rv3873 (SEQ ID No 16, PPE), Rv3874 (SEQ ID No 17, CFP-10), Rv3875 (SEQID No 18, ESAT-6), Rv3876 (SEQ ID No 19), Rv3877 (SEQ ID No 20), Rv3878(SEQ ID No 21), Rv3879 (SEQ ID No 22), Rv3880 (SEQ ID No 23), Rv3881(SEQ ID No 24), Rv3882 (SEQ ID No 25), Rv3883 (SEQ ID No 26), Rv3884(SEQ ID No 27) and Rv3885 (SEQ ID No 28).
 31. A cosmid or a plasmidcomprising a portion of DNA originating from Mycobacterium tuberculosisor any virulent member of the Mycobacterium tuberculosis complex (M.africanum, M. bovis, M. canettii), said portion of DNA comprising atleast one, two, three or more gene(s) selected from Rv3867 (SEQ ID No10), Rv3868 (SEQ ID No 11), Rv3869 (SEQ ID No 12), Rv3870 (SEQ ID No13), Rv3871 (SEQ ID No 14), Rv3872 (SEQ ID No 15, mycobacterial PE),Rv3873 (SEQ ID No 16, PPE), Rv3874 (SEQ ID No 17, CFP-10), Rv3875 (SEQID No 18, ESAT-6), Rv3876 (SEQ ID No 19) and Rv3877 (SEQ ID No 20). 32.A cosmid or a plasmid comprising a portion of DNA originating fromMycobacterium tuberculosis or any virulent member of the Mycobacteriumtuberculosis complex (M. africanum, M. bovis, M. canettii), said portionof DNA comprising at least one gene selected from Rv3871 (SEQ ID No 14),Rv3872 (SEQ ID No 15, mycobacterial PE), Rv3873 (SEQ ID No 16, PPE),Rv3874 (SEQ ID No 17, CFP-10), Rv3875 (SEQ ID No 18, ESAT-6) and Rv3876(SEQ ID No 12).
 33. A cosmid or a plasmid according to any of claims 30to 32 comprising Rv3874 encoding CFP-10, Rv3875 encoding ESAT-6 or bothor a part of them.
 34. A cosmid or a plasmid according to any of claims30 to 33 comprising a mutated gene selected among Rv3861 to Rv3885. 35.A cosmid or a plasmid according to claim 30 which comprises at leastfour genes selected from Rv3861 (SEQ ID No 4), Rv3862 (SEQ ID No 5),Rv3863 (SEQ ID No 6), Rv3864 (SEQ ID No 7), Rv3865 (SEQ ID No 8), Rv3866(SEQ ID No 9), Rv3867 (SEQ ID No 10), Rv3868 (SEQ ID No 11), Rv3869 (SEQID No 12), Rv3870 (SEQ ID No 13), Rv3871 (SEQ ID No 14), Rv3872 (SEQ IDNo 15, mycobacterial PE), Rv3873 (SEQ ID No 16, PPE), Rv3874 (SEQ ID No17, CFP-10), Rv3875 (SEQ ID No 18, ESAT-6), Rv3876 (SEQ ID No 19),Rv3877 (SEQ ID No 20), Rv3878 (SEQ ID No 21), Rv3879 (SEQ ID No 22),Rv3880 (SEQ ID No 23), Rv3881 (SEQ ID No 24), Rv3882 (SEQ ID No 25),Rv3883 (SEQ ID No 26), Rv3884 (SEQ ID No 27) and Rv3885 (SEQ ID No 28),provided that it comprises Rv3874 (SEQ ID No 17, CFP-10) and/or Rv3875(SEQ ID No 18, ESAT-6)).
 36. A cosmid or a plasmid according to claim 30which comprises at least Rv3871 (SEQ ID No 14), Rv3875 (SEQ ID No 18,ESAT-6) and Rv3876 (SEQ ID No 19)).
 37. A cosmid or a plasmid accordingto claim 30 which comprises at least Rv3871 (SEQ ID No 14), Rv3875 (SEQID No 18, ESAT-6) and Rv3877 (SEQ ID No 20).
 38. A cosmid or a plasmidaccording to claim 30 comprising at least Rv3871 (SEQ ID No 14), Rv3875(SEQ ID No 18, ESAT-6), Rv3876 (SEQ ID No 19) and Rv3877 (SEQ ID No 20).39. A cosmid or a plasmid according to one of claims 36 to 38 whichfurther comprises Rv3872 (SEQ ID No 15, mycobacterial PE) Rv3873 (SEQ IDNo 16, PPE) Rv3874 (SEQ ID No 17, CFP-10).
 40. A cosmid or a plasmidaccording to one of claims 36 to 38 which further comprises at least onetwo, three or four gene(s) selected from Rv3861 (SEQ ID No 4), Rv3862(SEQ ID No 5), Rv3863 (SEQ ID No 6), Rv3864 (SEQ ID No 7), Rv3865 (SEQID No 8), Rv3866 (SEQ ID No 9), Rv3867 (SEQ ID No 10), Rv3868 (SEQ ID No11), Rv3869 (SEQ ID No 12), Rv3870 (SEQ ID No 13), Rv3878 (SEQ ID No21), Rv3879 (SEQ ID No 22), Rv3880 (SEQ ID No 23), Rv3881 (SEQ ID No24), Rv3882 (SEQ ID No 25), Rv3883 (SEQ ID No 26), Rv3884 (SEQ ID No 27)and Rv3885 (SEQ ID No 28).
 41. A cosmid herein referred as RD1-2F9 andRD1-AP34 contained in the E. coli strains deposited at the CNCM underthe accession number I-2831 and I-2832 respectively.
 42. Use of a cosmidor a plasmid according to one of claims 30 to 41 for transforming M.bovis BCG or M. microti.
 43. A pharmaceutical composition comprising astrain according to one of claims 1 to 27 and a pharmaceuticallyacceptable carrier.
 44. A pharmaceutical composition according to claim40 containing suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the livingvaccine into preparations which can be used pharmaceutically.
 45. Apharmaceutical composition according to claim 40 or 41 which is suitablefor intravenous or subcutaneous administration.
 46. A vaccine comprisinga strain according to one of claims 1 to 28 and a suitable carrier. 47.A product comprising a strain according to one of claims 1 to 28 and atleast one protein selected from ESAT-6 and CFP-10 or epitope derivedthereof for a separate, simultaneous or sequential use for treatingtuberculosis.
 48. The use of a strain according to one of claims 1 to 28for preparing a medicament or a vaccine for preventing or treatingtuberculosis.
 49. The use of a strain according to one of claims 1 to 28as an adjuvant/immunomodulator for preparing a medicament for thetreatment of superficial bladder cancer.
 50. A method for theidentification at the species level of members of the M. tuberculosiscomplex by means of markers for RD1^(mic) and RD5^(mic) as moleculardiagnostic test.
 51. A method according to claim 50 comprising the useof a primer selected from: primer esat-6F GTCACGTCCATTCATTCCCT, (SEQ IDNo 32) primer esat-6R ATCCCAGTGACGTTGCCTT), (SEQ ID No 33) primerRD1^(mic) flanking region F GCAGTGCAAAGGTGCAGATA, (SEQ ID No 34) primerRD1^(mic) flanking region R GATTGAGACACTTGCCACGA, (SEQ ID No 35) primerRD5^(mic) flanking region F GAATGCCGACGTCATATCG, (SEQ ID No 39) primerRD5^(mic) flanking region R CGGCCACTGAGTTCGATTAT (SEQ ID No 40)

and the complementary sequences of said primers.
 52. A diagnostic kitfor the identification at the species level of members of the M.tuberculosis complex comprising DNA probes and primers specificallyhybridizing to a DNA portion of the RD1 or RD5 region of M.tuberculosis, more particularly probes hybridizing under stringentconditions to a gene selected from Rv3871 (SEQ ID No 14), Rv3872 (SEQ IDNo 15, mycobacterial PE), Rv3873 (SEQ ID No 16, PPE), Rv3874 (SEQ ID No17, CFP-10), Rv3875 (SEQ ID No 18, ESAT-6), Rv3876 (SEQ ID No 19) andRv3877 (SEQ ID No 20), preferably CFP-10 and ESAT-6.
 53. A diagnostickit according to claim 52 comprising a probe or primer selected from:primer esat-6F GTCACGTCCATTCATTCCCT, (SEQ ID No 32) primer esat-6RATCCCAGTGACGTTGCCTT), (SEQ ID No 33) primer RD1^(mic) flanking region FGCAGTGCAAAGGTGCAGATA, (SEQ ID No 34) primer RD1^(mic) flanking region RGATTGAGACACTTGCCACGA, (SEQ ID No 35) primer RD5^(mic) flanking region FGAATGCCGACGTCATATCG, (SEQ ID No 39) primer RD5^(mic) flanking region RCGGCCACTGAGTTCGATTAT (SEQ ID No 40)

and the complementary sequences of said primers.
 54. A diagnostic kitfor the identification at the species level of members of the M.tuberculosis complex comprising at least one, two, three or moreantibodies directed to mycobacterial PE, PPE, CFP-10, ESAT-6.
 55. Adiagnostic kit according to claim 54 wherein it comprises antibodiesdirected to CFP-10 and ESAT-6.
 56. Virulence markers associated with RD1and/or RD5 regions of the genome of M. tuberculosis or a part of theseregions.
 57. The use of a strain according to one of claims 1 to 28 as acarrier for the expression of a molecule or an heterologous antigen thatare of therapeutic or prophylactic interest.
 58. A purified nucleic acidcorresponding to the Mycobacterium DNA inserted in a cosmid according toany of claims 30 to
 41. 59. The purified nucleic acid according to claim58 which corresponds to the insert of cosmid RD1-2F9 or cosmid RD1-AP34.